Low shear microfluidic devices and methods of use and manufacturing thereof

ABSTRACT

Provided herein relates to systems and methods for producing and using a body having a central channel separated by one or more membranes. The membrane(s) are configured to divide the central channel into at least one mesochannel and at least one microchannel. The height of the mesochannel is substantially greater than the height of the microchannel. A gaseous fluid can be applied through the mesochannel while a liquid fluid flowing through the microchannel. The systems and methods described herein can be used for various applications, including, e.g., growth and differentiation of primary cells such as human lung cells, as well as any other cells requiring low shear and/also stratified structures, or simulation of a microenvironment in living tissues and/or organs (to model physiology or disease states, and/or to identify therapeutic agents and/or vaccines). The systems and methods can also permit co-culture with one or more different cell types.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is the U.S. national stage of International ApplicationNo. PCT/US2014/071611, filed on Dec. 19, 2014, which claims the benefitunder 35 U.S.C. § 119(e) of and priority to U.S. Provisional ApplicationNo. 61/919,193, filed on Dec. 20, 2013, each of which is incorporatedherein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.W911NF-12-2-0036 awarded by the DARPA. The government has certain rightsin the invention.

TECHNICAL FIELD

The present disclosure relates generally to microfluidic devices andmethods of use and manufacturing thereof. In some embodiments, themicrofluidic devices can be used for culture and/or support of livingcells such as mammalian cells, insect cells, plant cells, and microbialcells.

BACKGROUND

Mechanical forces—pushes, pulls, tensions, compressions—are importantregulators of cell development and behavior. Tensegrity provides thestructure that determines how these physical forces are distributedinside a cell or tissue, and how and where they exert their influence.

In the human body, most cells are constantly subjected to mechanicalforces, such as tension or compression. Application of mechanical strainto cells in culture simulates the in vivo environment, causing dramaticmorphologic changes and biomechanical responses in the cells. Both longand short term changes occur when cells are mechanically loaded inculture, such as alterations in the rate and amount of DNA or RNAsynthesis or degradation, protein expression and secretion, the rate ofcell division and alignment, changes in energy metabolism, changes inrates of macromolecular synthesis or degradation, and other changes inbiochemistry and bioenergetics.

Every cell has an internal scaffolding, or cytoskeleton, a latticeformed from molecular “struts and wires”. The “wires” are acrisscrossing network of fine cables, known as microfilaments, whichstretch from the cell membrane to the nucleus, exerting an inward pull.Opposing the pull are microtubules, the thicker compression-bearing“struts” of the cytoskeleton, and specialized receptor molecules on thecell's outer membrane that anchor the cell to the extracellular matrix,the fibrous substance that holds groups of cells together. This balanceof forces is the hallmark of tensegrity.

Tissues are built from groups of cells, like eggs sitting on the “eggcarton” of the extracellular matrix. The receptor molecules anchoringcells to the matrix, known as integrins, connect the cells to the widerworld. Mechanical force on a tissue is felt first by integrins at theseanchoring points, and then is carried by the cytoskeleton to regionsdeep inside each cell. Inside the cell, the force might vibrate orchange the shape of a protein molecule, triggering a biochemicalreaction, or tug on a chromosome in the nucleus, activating a gene.

Cells also can be said to have “tone,” just like muscles, because of theconstant pull of the cytoskeletal filaments. Much like a stretchedviolin string produces different sounds when force is applied atdifferent points along its length, the cell processes chemical signalsdifferently depending on how much it is distorted.

A growth factor will have different effects depending on how much thecell is stretched. Cells that are stretched and flattened, like those inthe surfaces of wounds, tend to grow and multiply, whereas roundedcells, cramped by overly crowded conditions, switch on a “suicide”program and die. In contrast, cells that are neither stretched norretracted carry on with their intended functions.

Another tenet of cellular tensegrity is that physical location matters.When regulatory molecules float around loose inside the cell, theiractivities are little affected by mechanical forces that act on the cellas a whole. But when the regulatory molecules are attached to thecytoskeleton, they become part of the larger network, and are in aposition to influence cellular decision-making. Many regulatory andsignaling molecules are anchored on the cytoskeleton at the cell'ssurface membrane, in spots known as adhesion sites, where integrinscluster. These prime locations are key signal-processing centers, likenodes on a computer network, where neighboring molecules can receivemechanical information from the outside world and exchange signals.

Thus, assessing the full effects of drugs, drug delivery vehicles,nanodiagnostics or therapies or environmental stressors, such asparticles, gases, and toxins, in a physiological environment requiresnot only a study of the cell-cell and cell-chemical interactions, butalso a study of how these interactions are affected by physiologicalmechanical forces in both healthy tissues and tissues affected withdiseases.

Methods of altering the mechanical environment or response of cells inculture have included wounding cells by scraping a monolayer, applyingmagnetic or electric fields, or by applying static or cyclic tension orcompression with a screw device, hydraulic pressure, or weights directlyto the cultured cells. Shear stress has also been induced by subjectingthe cells to fluid flow. However, few of these procedures have allowedfor quantitation of the applied strains or provided regulation toachieve a broad reproducible range of cyclic deformations within aculture microenvironment that maintains physiologically relevanttissue-tissue interactions.

Living organs are three-dimensional vascularized structures composed oftwo or more closely apposed tissues that function collectively andtransport materials, cells and information across tissue-tissueinterfaces in the presence of dynamic mechanical forces, such as fluidshear and mechanical strain. Creation of microdevices containing livingcells that recreate these physiological tissue-tissue interfaces andpermit fluid flow and dynamic mechanical distortion would have greatvalue for study of complex organ functions, e.g., immune celltrafficking, nutrient absorption, infection, oxygen and carbon dioxideexchange, etc., and for drug screening, toxicology, diagnostics andtherapeutics.

A major challenge lies in the lack of experimental tools that canpromote assembly of multi-cellular and multi-tissue organ-likestructures that exhibit the key structural organization, physiologicalfunctions, and physiological or pathological mechanical activity of thelung alveolar-capillary unit, which normally undergoes repeatedexpansion and contraction during each respiratory cycle. This limitationcould be overcome if it were possible to regenerate this organ-levelstructure and recreate its physiological mechanical microenvironment invitro. However, this has not been fully accomplished.

What is needed is a organ mimic device capable of being used in vitro orin vivo which performs tissue-tissue related functions and which alsoallows cells to naturally organize in the device in response to not onlychemical but also mechanical forces and allows the study of cellbehavior through a membrane which mimics tissue-tissue physiology.

SUMMARY

The existing transwell technology has been widely used to grow anddifferentiate human cells. The invention is directed to, inter alia, aplatform and method for growth and differentiation of human cells in amicrofluidic environment. Previously developed organ-on-chip devices aredescribed in the International Patent Application Nos. PCT/US2009/050830and PCT/US2012/026934, which are incorporated herein by reference intheir entireties. In accordance with one embodiment of the invention, amicrofluidic device can include a top mesochannel with a channel heightof about 1 mm and a bottom microchannel with a channel height of about100 μm. By increasing the height of at least a length portion of the topchannel within the device (e.g., the length portion where cells aredesired to grow to form a stratified/pseudostratified or 3-dimensionalstructure), the device can provide at least a length portion of the topchannel with a reduced stress environment and increased overhead spacefor growth of cells that require low shear and/or more space to form astratified, pseudostratified, or three-dimensional tissue structure. Inone embodiment, airway epithelial cells (e.g., small airway and/or largeairway epithelial cells) can be cultured on the surface of the membranefacing the mesochannel and can differentiate into terminallydifferentiated ciliated and mucous-secreting (goblet) cells. Other cellsthat are desired to be cultured in a higher top channel include, but arenot limited to, heart cells, gut cells/intestinal cells, liver cells,skin cells, and kidney cells (e.g., glomerular cells). For example,intestinal epithelial cells can be cultured on the surface of themembrane facing the mesochannel and form three-dimensional intestinalvilli. In some embodiments, animal cells, insect cells, and plant cellscan also be used in the devices described herein.

System and method comprises a body having a central channel separated byone or more membranes. The membranes divide the central channel into twoor more closely apposed parallel central sub-channels (mesochannels andmicrochannels), wherein one or more first fluids (e.g., gaseous orliquid fluid) can be applied through at least one mesochannel and one ormore second fluids (e.g., liquid fluid) can be applied through one ormore microchannels. The surfaces of each membrane can be treated orotherwise coated with cell adhesive molecules to support the attachmentof cells and/or promote their organization into tissues on the upperand/or lower surface of each membrane, thereby creating one or moretissue-tissue interfaces separated by one or more membranes between theadjacent parallel fluid channels. The membrane can be porous (e.g.,permeable or selectively permeable), non-porous (e.g., non-permeable),rigid, flexible, elastic, or any combination thereof. In someembodiments, the membrane can be porous, e.g., allowingexchange/transport of fluids (e.g., gas and/or liquids), passage ofmolecules such as nutrients, cytokines and/or chemokines, celltransmigration, or any combinations thereof. In some embodiments, themembrane can be non-porous. Fluid pressure, flow characteristics andchannel geometry can be varied to apply a desired fluid (e.g., airand/or liquid) shear stress to one or both tissue layers.

In some embodiments, an air-liquid interface can be established in thedevices described herein to mimic a physiological microenvironment,e.g., an airway, thus permitting cells to behave more like cells invivo, e.g., differentiation of airway epithelial cells to ciliatedand/or mucus-secreting cells to form a stratified structure. In someembodiments, a unidirectional or a bidirectional flow of gas (e.g., air)can be induced in the mesochannel by adapting one end of the mesochannelto engage to a gas-flow modulation device.

In some embodiments, the membrane of the device can be modulated oractuated to deform in a manner (e.g., stretching, retracting,compressing, twisting and/or waving) that simulates a physiologicalstrain experienced by the cells in its native microenvironment, e.g.,during breathing, peristalsis, and/or heart beating. In some embodimentswhere operating channels are adjacent to the central channel, a positiveor negative pressure can be applied to the operating channels, which canin turn create a pressure differential that causes the membrane to, forexample, selectively stretch and retract in response to the pressuredifferential, thereby further physiologically simulating mechanicalforce of a living tissue-tissue interface. For example, in someembodiments, a combination of culturing intestinal cells in a tallermesochannel for increased overhead space and lower liquid shear stress,and exposure of the cells to physiological peristalsis-like motionsinduced by cyclically stretching and retracting the membrane can inducehuman intestinal cells to form a three-dimensional villus structure.

In some embodiments, the devices described herein can permit two or moredifferent cell types cultured in the same channel (e.g., mesochannel ormicrochannel), and/or in different channels (e.g., at least one celltype in the mesochannel and at least one cell type in the microchannel;or a first cell type in a first mesochannel and a second cell type in asecond mesochannel). For example, tissue-specific epithelial cells canbe cultured on one side of the membrane facing the mesochannel, whileblood vessel-associated cells can be cultured on the other side facingthe microchannel. By way of example only, in some embodiments, microbialcells, e.g., healthy or diseased microbial flora, can be cultured withthe intestinal epithelial cells in the mesochannel to mimic thephysiological microenvironment of a normal or diseased intestine invivo.

In some embodiments, immune cells can be added to a liquid fluid presentin the microchannel. The liquid fluid in the microchannel can be staticor flowing through the microchannel continuously, cyclically, and/orintermittently. Recruitment of immune cells to the membrane and/ortissue-specific cells can be determined to provide a measure of immuneresponse when the simulated physiological microenvironment is stimulatedwith an agent or a cytokine described herein. The ability to introduceimmune cells in the device described herein and measure response of theimmune cells (e.g., immune cell recruitment, maturation, activation,cell killing, and/or drainage) permits development of a more accuratetissue-specific disease model that takes into account of immune responseas is typically activated in vivo when a subject is afflicted with adisease (except in a subject who is immunocompromised).

The ability of the devices described herein to recapitulate aphysiological microenvironment and/or function can provide an in vitromodel versatile for various applications, e.g., but not limited to,generation of cells corresponding to a physiological endpoint asdescribed herein; modeling a tissue-specific physiological condition(e.g., but not limited to normal and disease states); determination oftransmissibility of airborne pathogens, development of mucosal immunityplatform; identification of therapeutic agents and vaccines; and anycombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a block diagram of a system employing an exampleorgan mimic device in accordance with an embodiment.

FIG. 2A illustrates a perspective view of an organ mimic device inaccordance with an embodiment.

FIG. 2B illustrates an exploded view of the organ mimic device inaccordance with an embodiment.

FIG. 2C illustrative perspective views of organ mimic devices withdifferent positions of inlet and outlet ports. The top panel illustratesthat the inlet and outlet ports are positioned on a top surface of aportion of the device described herein. The bottom panel illustratesthat the inlet and outlet ports are positioned on the lateral side of aportion of the device described herein.

FIG. 2D illustrates a diagrammatic view of a cell-cell interface regionof the device in accordance with an embodiment.

FIGS. 2E-2F illustrate cross sectional views of a top body portion and abottom body portion of the device in accordance with an embodiment,respectively.

FIG. 2G illustrates a cross-sectional view of a device in accordancewith an embodiment.

FIG. 2H illustrate a top view of a device in accordance with theembodiment described in FIG. 2D.

FIG. 3A is a schematic of a photolithography process used to fabricate abottom body portion of the device comprising a microchannel inaccordance with an embodiment.

FIG. 3B is a schematic of a stereolithographic process used to fabricatea top body portion of the device comprising a mesochannel in accordancewith an embodiment.

FIG. 3C is a set of images showing a CAD model of tall channel “wafer”(left panel) and a stereolithographic thermoplastic reconstruction(right panel).

FIGS. 4A-4C illustrate different methods of forming a fluidic sealbetween the membrane and the top and bottom body portions of the devicein accordance with an embodiment. FIG. 4A illustrates3-aminopropyl-triethoxysilane (APTES) bonding procedure adopted fromAran et al. (2010). FIG. 4B shows membrane clamping between the PDMSslabs utilizing the membrane-PDMS surface area. FIG. 4C illustrates amembrane sealed between the two pieces of PDMS slabs by plasma bondbetween the PDMS slabs to form a seal.

FIGS. 5A-5G illustrate an exemplary method of differentiating humanprimary airway (or bronchial) epithelial cells in a device in accordancewith an embodiment, and experimental data resulting therefrom. FIG. 5Ais a schematic diagram illustrating an example method to differentiatehuman bronchial epithelial cells in a device according to an embodiment.Primary small airway or bronchial epithelial cells were seeded on themembrane in the upper mesochannel (an “airway lumen” channel). The cellswere then cultured in a submerged condition by introducing a static orflowing culture medium into both the mesochannel and the microchanneluntil the cells reached full confluence. Then, an air-liquid interfacewas established by removing the culture medium from the mesochannelthrough its outlet. The primary airway epithelial cells differentiatedafter about 3-4 weeks of culture in the device at the air-liquidinterface. FIG. 5B is a diagrammatic view of human differentiatedbronchial epithelium grown in a mesochannel separated from the bottommicrochannel (a “blood vessel” channel) by a porous membrane. FIG. 5Cillustrates morphology of the cells post-seeding and at the time whenthe air-liquid interface (ALI) was set up. FIG. 5D is a set ofimmunofluorescence images showing formation of a primary small airwayepithelium on the membrane. Tight junction proteins (e.g., TJP-1 and/orZO-1) were detected to indicate a functional tight junction barrierformed by the formed epithelium. FIG. 5E is a set of immunofluorescenceand SEM images showing differentiation of the airway epithelial cells tociliated cells. FIG. 5F shows a 3D view of differentiated epithelialprimary cells (cilia: detected by beta-tubulin IV; and mucus secretion:detected by Muc5AC) in the device described herein. FIG. 5G showsrepresentative images of ciliated cells along the length of themesochannel of the device described herein. A uniform distribution ofabundant cilia beating after about 3 weeks of culturing at an air-liquidinterface is a hallmark of epithelial differentiation in vivo.

FIGS. 6A-6B show cell viability data directed to cultures of primaryhuman small airway epithelial cells in a device according to anembodiment and in a transwell. FIG. 6A is a bar graph comparing thecytotoxicity data (based on LDH release from the cells) of culturingprimary human small airway epithelial cells in a microfluidic device (tomimic a small airway) with the cells cultured in a transwell. FIG. 6B isa microscopic image showing the healthy small airway epithelial cellsforming an intact epithelium in the device described herein.

FIGS. 7A-7D show experimental data indicating differentiation of humanbronchial epithelial cells in a mesochannel of a device in accordancewith an embodiment. FIG. 7A is a set of florescent images showing thathuman bronchial epithelial cells grown in the mesochannel for about 4weeks exhibited typical differentiation markers of an human bronchialepithelium in vivo. Beta-tubulin can be used as a marker to detectciliated cells (upper left panel). The orthogonal section (lower panel)shows that cilia are localized on the apical side of the cultures asobserved in vivo. The right panel is an image showing a 3Dreconstruction of the epithelium. FIG. 7B is a set of fluorescent (leftpanel) and SEM (right panel) images showing that human bronchialepithelial cell cultures in a mesochannel of a device described hereindisplay ciliated and goblet cells after about 3 weeks of culturing at anair-liquid interface. The SEM image shows fully formed cilia. FIG. 7C isa set of fluorescent images showing presence of tight junctions, asindicated by ZO-1 and phalloidin staining, formed between thedifferentiated human bronchial cells cultured in a mesochannel of adevice according to an embodiment. FIG. 7D is a graph showing thebarrier function of the differentiated epithelium formed in themesochannel of a device described herein. The barrier function wasevaluated by adding fluorescently-labeled large molecules (e.g.,inulin-FITC) into the fluid introduced into the mesochannel. Thedifferentiated epithelium prevents inulin to cross from the mesochannelto the microchannel, indicating that the epithelium forms a functionalbarrier.

FIG. 8 is a set of images showing co-culture of human primary airwayepithelial cells on one side of a porous membrane facing the mesochannelwith the endothelial cells cultured on another side of the porousmembrane facing the microchannel.

FIG. 9 is a schematic diagram illustrating an example experimentaldesign for a neutrophil recruitment assay. Primary small airwayepithelial cells were seeded on the membrane in the mesochannel (an“airway lumen” channel) for differentiation into ciliated and gobletcells following the differentiation method as described in FIG. 5A. Oncethe cells are differentiated, endothelial cells can be seeded on anothersurface of the porous membrane forming a coculture. The cells can thenbe contacted with an agent and neutrophil recruitment can be determinedby measuring attachment of neutrophils to the endothelial monolayer.

FIGS. 10A-10C illustrate an exemplary method of evaluating neutrophilrecruitment in response to inflammation induced by challenging thedifferentiated human primary airway (or bronchial) epithelial cells in adevice with a pro-inflammatory factor, e.g., TNFα, and experimental dataresulting therefrom. FIG. 10A is a schematic diagram illustrating anexample method to evaluate neutrophil recruitment in response to astimulus using a device according to an embodiment. Primary small airwayepithelial cells were seeded on the membrane in the mesochannel (an“airway lumen” channel) for differentiation into ciliated and/ormucus-secreting cells following the differentiation method as describedin FIG. 5A. Upon differentiation of the airway epithelial cells, anothersurface of the membrane (facing the microchannel, the “blood vessel”channel) could be seeded with or without endothelial cells. Thedifferentiated cells in the mesochannel were then challenged with apro-inflammatory factor, e.g., TNF-α. A fluid comprising humanneutrophils was then flowed through the “blood vessel” channel todetermine effects of TNF-α-induced inflammation on neutrophilrecruitment. FIG. 10B includes a data graph showing quantification ofneutrophils attached to differentiated epithelial cells cultured in themesochannel with or without the treatment of TNFα (right panel). Thequantification is based on counting the number of attached neutrophil inthe fluorescent images (left panel) taken by microscopy imaging. FIG.10C is a snapshot image showing neutrophil flowing through the “bloodvessel” channel at a specific time point.

FIGS. 11A-11D illustrate an exemplary method of evaluating an infectionresponse of differentiated small airway epithelial cells and optionallyimmune cells in a device in accordance with an embodiment, andexperimental data resulting therefrom. FIG. 11A is a schematic diagramillustrating an example method to evaluate an infection response in thedevice. Primary small airway epithelial cells were seeded on themembrane in the mesochannel (an “airway lumen” channel) fordifferentiation into ciliated and/or mucus-secreting cells following thedifferentiation method as described in FIG. 5A. Upon differentiation ofthe airway epithelial cells, another surface of the membrane (facing themicrochannel, the “blood vessel” channel) could be seeded with orwithout endothelial cells. The differentiated cells in the mesochannelwere then challenged with a toll-like receptor 3 (TLR-3) ligand poly I:Cto induce inflammation. A fluid comprising immune cells (e.g., humanmonocytes) was introduced into the “blood vessel” channel, either with astatic fluid or a flowing fluid, to determine effects of TLR-3-inducedinflammation on cytokine/chemokine profiles of the differentiated cellsand/or recruitment of immune cells (e.g., monocytes and/or neutrophils).FIG. 11B is a set of bar graphs showing that TLR-3 activation (flu-likesituation) stimulates release of chemokines (e.g., monocytechemoattractants and neutrophil chemoattractants) by the differentiatedairway epithelial cells in the device. FIG. 11C is a set of data showingquantification of monocytes attached to the TLR-3 activateddifferentiated epithelial cells in the device and the associatedfluorescent images. FIG. 11D is a graph showing gene expression ofdifferentiated epithelial cells after treatment with or without a TLR-3ligand poly I:C.

FIGS. 12A-12F illustrate an exemplary method of evaluating an effect ofdifferent agents on differentiated small airway epithelial cells andoptionally immune cells during an infection in a device in accordancewith an embodiment, and experimental data resulting therefrom. FIG. 12Ais a schematic diagram illustrating an example method to evaluate aneffect of different agents during an infection simulated in the device.Primary human epithelial cells from chronic obstructive pulmonarydisease (COPD) patients (obtained from a commercial vendor) were seededon the membrane in the mesochannel (an “airway lumen” channel) fordifferentiation into ciliated and/or mucus-secreting cells following thedifferentiation method as described in FIG. 5A. Upon differentiation ofthe COPD epithelial cells, another surface of the membrane (facing themicrochannel, the “blood vessel” channel) could be seeded with orwithout endothelial cells. The cells in the device were then starvedusing basal medium, followed by treatment with different agents (e.g.,DMSO as a control, budesonide, and BRD4 inhibitor compounds 1 and 2obtained from a pharmaceutical company). The agents were delivered tothe differentiated epithelial cells via diffusion from the “bloodvessel” channel. The pre-treated differentiated COPD epithelial cellswere then challenged with TLR-3 ligand poly I:C (e.g., about 10 μg/mLdelivered as an aerosol flowing into the mesochannel) to stimulate TLR-3and mimic viral infection. Secreted cytokines and chemokines from thedifferentiated COPD epithelial cells were quantified in the flow-throughof the “blood vessel” channel and/or from the apical wash of the “airwaylumen” channel. In some embodiments, a fluid comprising immune cells(e.g., human monocytes) was introduced into the “blood vessel” channel,either with a static fluid or a flowing fluid, to determine effects ofTLR-3-induced inflammation on recruitment of immune cells (e.g.,monocytes and/or neutrophils). FIG. 12B is a set of graphs showingproduction of representative cytokines and chemokines (e.g., monocytechemoattractants and neutrophil chemoattractants) by the differentiatedCOPD epithelial cells (pretreated with different agents prior toexposure to a TLR-3 ligand poly I:C) and released into the “bloodvessel” channel. It indicates that compound 2 is more potent thancompound 1 in reducing cytokine/chemokine secretion in response to thesimulated viral infection. FIG. 12C is a table summarizing effects ofdifferent agents on release of some of the cytokines/chemokines from thedifferentiated COPD epithelial cells into the “blood vessel” channel.FIG. 12D is a table summarizing effects of different agents on secretionof some of the cytokines/chemokines by the differentiated COPDepithelial cells into the “airway lumen” channel. FIG. 12E is a graphshowing gene expression of differentiated COPD epithelial cellspretreated with different agents prior to exposure to a TLR-3 ligandpoly I:C. FIG. 12F is a graph showing quantification of neutrophilattachment to TLR-3 stimulated differentiated COPD epithelial cells inthe device described herein. The graph shows that compound 2 is morepotent in reducing neutrophil adhesion, whereas compound 1 did not havesuch effect, and such result is consistent with and validates thepharmaceutical company's in-house data on potency of compound 2 inreducing inflammation.

FIGS. 13A-13D are photographs of an example experimental set-up orgas-flow modulation device to simulate respiration/breathing in a devicedescribed herein. FIG. 13A is a photograph showing an overview of asystem for simulating respiration/breathing through an airway of a lung.The system comprises a device according to one embodiment, wherein themesochannel of the device is adaptably connected to a ventilator (forair-flow generation); and optionally an optical device (e.g., amicroscope) for monitoring the cells. The breathing dynamics inside thedevice can be controlled and/or monitored using a pre-programmedcomputer. FIG. 13B shows an example method to provide a bi-directionalflow of air through the mesochannel of the device described herein. Thetop panel is a diagrammatic top view of a small airway-on-a-chipindicating the inlet and outlet of the mesochannel (the “airway lumen”channel). The bottom panel shows that using a small animal ventilatorand other equipment, rhythmic airflow can be introduced into themesochannel (e.g., 15 breaths per min; tidal volume of about 100μL/breath). In addition, the outlet of the mesochannel is adaptablyconnected to a gas-flow modulation device (e.g., an inflatable chambersuch as a balloon) to facilitate expiration of air out of the device(due to the chamber material's elasticity and compliance). FIG. 13C is aphotograph showing a balloon-located at the outlet of the mesochannel(the “airway lumen” channel)—expands due to accumulation of air flowninto the device through the inlet of the mesochannel and contracts topush the air back due to its elasticity and compliance. FIG. 13D is aset of photographs showing an alternative embodiment of a gas-flowmodulation device, which is a drum comprising a flexible diaphragm. Asthe ventilator pushes the air in (inspiratory flow) through the inlet ofthe mesochannel, the drum diaphragm moves outward (inflates) and inward(deflates) to accumulate and expel the air, respectively.

FIGS. 14A-14B are experimental data showing simulation of respiration ina device according to one embodiment. One end of the mesochannel (the“airway lumen” channel) of the device was adaptably connected to, e.g.,a small animal ventilator and attached equipment that can adjustpressure and volume of air, in order to generate air flow. Air was flownfrom the one end of the “airway lumen” channel, namely “mouth end” intothe device—that is “inspiratory flow.” The other end of the “airwaylumen” channel, known as “alveolar end” was adaptably connected to arubber balloon structure with compliance and elasticity to help forcingthe air out of the device—that is “expiratory airflow.” Theairflow/breathing was adjusted in a way to mimic breathing of a humansubject in the resting state at a small airway level˜15×(inspiration+expiration) cycles with tidal volume average of 100 μl,and can be adjusted to accommodate different breathings patterns and/ortidal volumes. About 24 hrs after breathing, ˜2 μm fluorescence (red)beads were added into the “airway lumen” channel, i.e., on top ofepithelial cells, and the movement of the fluorescent beads was followedby microscope. This set-up can be used to determine ciliary clearancerate. FIG. 14A is a set of snapshot images showing the movement of thefluorescent beads within the “airway lumen” channel of the device at aspecific time point. The left panel is directed to a control device thatdid not receive airflow and shows partially polarized beadmovements—i.e. some beads in one direction, a few in the oppositedirection. The right panel is directed to a device that received airflowfor about 24 hrs and shows more polarized bead movement towards the“mouth end.” FIG. 14B is a bar graph showing a higher ciliary clearancerate (determined by movement of the fluorescent beads) in the devicethat received airflow (breathing chip) than in the control devicewithout airflow (the non-breathing chip).

FIG. 15 is a schematic diagram showing an example system to evaluatetransmissibility of airborne pathogens. The system comprises a“pathogen-infected” small airway-on-a-chip and a “uninfected” smallairway-on-a-chip, wherein the mesochannel of the “pathogen-infected”small airway-on-a-chip is fluidically connected to the mesochannel ofthe “uninfected” small airway-on-a-chip. An inspiratory airflow isintroduced into the mesochannel of the pathogen-infected” smallairway-on-a-chip, and the output airflow is directed to the “uninfected”small airway-on-a-chip to determine airborne transmissibility.

FIG. 16 is a schematic diagram showing a cross-sectional view of adevice according to one embodiment that can be used to form a mucosalimmunity platform to study immune cell recruitment, maturation andactivation and drainage. Immune cells are introduced into the “bloodvessel” channel, either with a static fluid or a flowing fluid, andtheir behavior (e.g., trans-epithelial migration, maturation, activationand/or drainage back to the “blood vessel” channel) are monitored. Theplatform can be used to study role of airway mucosal surface in innateand adaptive immunity.

FIGS. 17A-17B are images showing squamous phenotype of bronchial cellculture in a device in accordance with an embodiment (FIG. 17A), andreversal of the squamous phenotype by addition of retinoic acid (FIG.17B).

FIG. 18 is a set of images showing morphology of human airway epithelialcells from asthmatic donors and normal donors cultured in a device inaccordance with one embodiment.

FIG. 19 is a photograph showing a system in which more than one devicesdescribed herein (e.g., 8-16 devices) can be fluidically connected toeach other and/or to fluid sources. The system can comprise an incubatorto provide a temperature-controlled environment for the devices.

FIG. 20 illustrates a system diagram employing more than one devicesdescribed herein, which are fluidically connected to each other and/orto fluid sources.

FIG. 21 illustrates integration of an inertial impactor into oneembodiment of a device described herein for aerosol delivery of anagent.

FIGS. 22A and 22B illustrate alternative embodiments of a devicedescribed herein. FIG. 22A illustrates a device comprising at least onemesochannel separated from at least two microchannels by a membrane.FIG. 22B illustrates a device comprising at least two mesochannelsseparated from at least one microchannel by a membrane.

FIG. 23 illustrates a top view of a device in accordance with theembodiment described in FIG. 2D.

FIGS. 24A-24B show transverse cross sectional views of a device withoperating channels according to some embodiments described herein. Inthese embodiments, the height of the operating channels is greater thanthe height of the mesochannel and/or the height of the microchannel. Asshown in the figures, the membrane is constructed to include a centralregion, wherein the central region includes the portion of the membraneseparating the mesochannel from the microchannel. In some embodiments,the membrane can be extended into the operating channel(s) andseparating the operating channel(s) into two or more compartments (asshown in the figures). In alternative embodiments, the operatingchannel(s) does not/do not contain any membrane separating the operatingchannel(s) into two or more compartments.

FIGS. 25A-25B are confocal images of well-differentiated normal andchronic obstructive pulmonary disease (COPD) epithelia followingair-liquid interface (ALI) induction in the device according to one ormore embodiments described herein. (FIG. 25A) Top panel shows primaryhealthy donor-derived epithelium. Bottom panel is an orthogonal sectionshowing apical cilia coverage in pseudostratified columnar epitheliaNote the apical localization of the cilia. (FIG. 25B) Top panel showsepithelial cells obtained from a COPD patient. Bottom panel is anorthogonal section showing apical cilia coverage in pseudostratifiedcolumnar epithelia (nucleic were counterstained with DAPI). In FIGS.25A-25B, ciliated cells were labeled fro β-tublin IV andmucous-producing goblet cells were stained with anti-MUC5AC antibody.Nucleic were counterstained with DAPI. Note the apical localization ofthe cilia. (Top panels) Scale bar, 20 μm. (Bottom panels) Scale bar, 20μm.

FIGS. 26A-26E are data graphs showing COPD disease phenotype can beestablished in the device according to one or more embodiments describedherein. FIG. 26A shows the mRNA levels of TLR 4 (left) and TLR3 (right)expression between healthy and COPD-derived epithelial cells that weregrown in the device (4 COPD donors and 6 healthy subjects). FIG. 26Bcompares IL-8 secretion between COPD and healthy epithelia after LPS(lipopolysaccharides) stimulation. FIG. 26C compares M-CSF secretionbetween COPD and healthy epithelia after poly (I:C)(polyinosinic:polycytidylic acid) stimulation. FIGS. 26D-26E show theexpression of cytokines IP-10 (FIG. 26D) and RANTES (FIG. 26E) inducedin both healthy donor and COPD epithelial cells upon stimulation withpoly(I:C) (4 donors for both groups per condition used).

FIGS. 27A-27C show establishment of a three-cell type microfluidicco-culture system comprising ciliated epithelium, endothelium andcirculating leukocytes. (FIG. 27A) Left panel: a 3D cross-sectionaldiagram of one embodiment of the devices described herein used tore-create post-capillary venules (major sites of leukocyte recruitmentand adhesion in vivo). Middle panel: a schematic diagram showing a3-cell type co-culture of epithelium, endothelium and neutrophils (allprimary cells). Right panel: vertical immunofluorescence cross-sectionof ciliated epithelium and endothelium bilayer on-device. Ciliated cellswere labeled for β-tubulin IV and endothelial cells were stained withanti-CD31/PECAM-1 antibody. Nuclei were counterstained with DAPI. Scalebar, 20 μm. (FIG. 27B) A series of time-lapse microscopic images showedcapture of a flowing neutrophil (not visible in the first panel fromleft but appears in the second panel; shown by the arrow head) toendothelium adjacent to a pre-adhered neutrophil (circles). Followinginitial attachment the neutrophil crawled over apical surface ofactivated endothelium and then firmly adhered (times indicated inseconds). Neutrophils and endothelial cells had been live stained withCellTracker Red and Calcein AM, respectively. (FIG. 27C) Bar graphsshowing E-selectin and VCAM1 mRNA levels in endothelia cells upontreatment of differentiated epithelial cells with or without poly (I:C).(3 devices per condition were used)

FIGS. 28A-28C show capability of determining drug efficacy on neutrophilcapture and adhesion and inflammation suppression in a small airwaymimicking device according to one or more embodiments described herein.(FIG. 28A) Representative immunofluorescence images showing adhesion ofrecruited neutrophils under three different conditions: (left) no drug;(middle) budesonide; (right) PFI-2. Neutrophils were stained withHoechst immediately prior to experiment to allow visualization andquantification. (FIG. 28B) Bar graph showing percentage change inneutrophil adhesion to activated endothelium as imaged in FIG. 28A. (n=3different donors per condition; 7-8 devices per condition from 4independent experiments; 4-5 distinct fields per chip). (FIG. 28C) A setof bar graphs showing levels of different cytokine secretion modulatedby the indicated drug or under no treatment. Cytokines measured include:neutrophil-attractant IL-8, GROα, and GM-CSF, monocyte-chemoattractantMCP-1, and acute inflammation associated cytokine IL-6. (n=3 donors percondition).

FIGS. 29A-29B are images showing human airway epithelial cellsdifferentiated into Clara cells in one or more embodiments of thedevices described herein. (FIG. 29A) Confocal microscopic top view imageof Clara cells stained for CC10 and ciliated cells labeled withβ-tubulin IV following well-differentiation of bronchiolar cells in thedevice. Scale bar, 10 μm. (FIG. 29B) Representative scanning electronmicrograph of differentiated bronchiolar cells grown in the device,showing the extensive ciliated cells coverage (“1” arrow), microvilli(“2” arrow) that normally indicates apical membrane of mucous-producinggoblet cells, and some dome-shaped structures that indicate Clara cells(“3” arrow). Scale bar, 10 μm.

FIGS. 30A-30D show induction of asthma-like phenotype in theairway-on-a-chip for assessment of drug efficacy. FIG. 30A is a set offluorescent images showing airway chips stimulated with IL-13 exhibit ahigher number of goblet cells (cells that produce mucus). FIG. 30B is abar graph showing quantification of globet cell coverage based on thefluorescent images (representative images shown in FIG. 30A). FIG. 30Cis a set of bar graphs showing secretion of G-CSF and GM-CSF by IL-13stimulated cells, as compared to cells without IL-13 stimulation. FIG.30D is a bar graph showing cilia beating frequency under indicatedconditions. In FIGS. 30B-30D, the “airway” devices were also used toassess the drug efficacy of Tofacitinib, a JAK inhibitor, by treatingthe IL-13 stimulated cells with Tofacitinib, and measuring eachphenotype as described above accordingly.

DETAILED DESCRIPTION OF THE INVENTION

Example embodiments of various aspects are described herein in thecontext of an organ simulating device and methods of use andmanufacturing thereof. In particular, one embodiment of the invention isdirected to, inter alia, an organ simulating device and methods of usesthereof for growth and differentiation of cells (e.g., cells thatrequire low shear and/or form a stratified structure or a 3-dimensionaltissue). In accordance with various aspects described herein, an organsimulating device comprises a first channel and a second channelseparated by a membrane, wherein the first channel (termed a“mesochannel” described herein) has a height sufficient to allow cellsto form a pseudostratified or stratified structure, or athree-dimensional tissue structure. In one embodiment, the height of themesochannel can be substantially greater than the height of the secondchannel (also referred to as the microchannel). In another embodiment,the height of the mesochannel can be substantially same as the height ofthe second channel.

For example, the inventors have demonstrated in one embodiment of thedevice described herein well-differentiation of primary human airwayepithelial cells (e.g., small airway epithelial cells) into ciliatedcells, mucous-secreting goblet cells, and Clara cells in apseudostratified structure, by culturing the airway epithelial cells atan air-liquid interface established within the device. In thisembodiment, the device comprises a mesochannel and a microchannel,wherein the mesochannel has a height (e.g., ˜1000 μm) that issubstantially higher than that of the microchannel (e.g., ˜100 μm). Themesochannel can be adapted to mimic an “airway lumen” channel and themicrochannel can be adapted to mimic a “blood vessel” channel. Forexample, to form an “airway lumen” channel, airway or bronchialepithelial cells are seeded on the membrane facing the upper mesochannel(the “airway lumen” channel) and the epithelial cells differentiateafter about 3-4 weeks of culture in the device at the air-liquidinterface.

In addition, unlike the existing open-top transwell system that has beenpreviously used to grow and differentiate human cells, but does notallow delivery of air (with a given volume, direction, and speed) on topof epithelial cells, the mesochannel (or an “airway lumen” channel) canbe adapted to form a closed top system, which allows airflow overdifferentiated epithelial cells to mimic breathing pattern and/orrhythm. For example, one end of the mesochannel can be adapted toconnect to a gas-flow modulation device (e.g., a reversibly expandableor inflatable chamber) that is adapted to provide a unidirectionaland/or a bi-directional flow of air in the mesochannel. Thus, air can bedelivered through the mesochannel (at a predefined volume, rate and/orspeed) into and out of the device to mimic respiration and/or permitaerosol delivery of compounds, chemicals and/or biologics. Further, thedirectionality of airflow in the mesochannel can also facilitatedirectional and rhythmic beating of the differentiated ciliated cellsgrown in the mesochannel, which can be in turn used to determinemucociliary clearance of a particle (e.g., debris, pathogens and/orparticulates) over the length of the mesochannel from one end toanother.

Additionally, the device can be used to determine recruitment of immunecells (e.g., but not limited to, CD8+ T cells, lymphocytes, monocytes,and/or neutrophils) from a static or flowing medium in the bottommicrochannel (or the “blood vessel” channel) to the membrane, or toblood vessel-associated cells (e.g., endothelial cells, fibroblasts,pericytes and/or smooth muscle cells) grown on another surface of themembrane facing the microchannel (or the “blood vessel” channel), bothof which can represent or model an inflammatory response (e.g., involvedin a respiratory disease or an infection). Thus, various embodiments ofthe devices described herein can be used to model and study respiratorydiseases (e.g., asthma, chronic obstructive pulmonary disease (COPD),pulmonary hypertension, cystic fibrosis, and any disease associated witha respiratory system including, e.g., nasal, trachea, bronchus, and/orairway), radiation-induced injury, and/or infectious disease (e.g.,viral or bacterial infection). These disease models can be in turn used,e.g., for drug screening, and/or study of pathophysiology of variousdiseases or disorders.

While in one embodiment, the device described herein is suitable forgrowth and differentiation of human lung cells including alveolar,airway, bronchial, tracheal and nasal epithelia, the device describedherein can also be used for other organs-on-a-chip requiring tallerchannel height to support optimal cell culture and/or formation ofmultiple cell layers or a three-dimensional tissue structure, forexample, including but not limited to Skin-on-a-Chip, Liver-on-a-Chip,Gut-on-a-Chip, Heart-on-a-Chip, Eye-on-a-Chip, and others. Accordingly,in some embodiments, the devices described herein can be used to modeldiseases other than respiratory diseases, e.g., but not limited to, skindisease, liver diseases, gastrointestinal diseases, heart diseases, andocular diseases.

Those of ordinary skill in the art will realize that the followingdescription is illustrative only and is not intended to be in any waylimiting. Other embodiments will readily suggest themselves to suchskilled persons having the benefit of this disclosure. Reference willnow be made in detail to implementations of the example embodiments asillustrated in the accompanying drawings. The same reference indicatorswill be used throughout the drawings and the following description torefer to the same or like items. It is understood that the phrase “anembodiment” encompasses more than one embodiment and is thus not limitedto only one embodiment for brevity's sake.

In accordance with this disclosure, the organ mimic device (alsoreferred to as “present device”) is preferably utilized in an overallsystem incorporating sensors, computers, displays and other computingequipment utilizing software, data components, process steps and/or datastructures. The components, process steps, and/or data structuresdescribed herein with respect to the computer system with which theorgan mimic device is employed can be implemented using various types ofoperating systems (e.g., Windows™, LINUX, UNIX, etc.) computingplatforms (e.g., Intel, AMD, ARM, etc.), computer programs, and/orgeneral purpose machines. In addition, those of ordinary skill in theart will recognize that devices of a less general purpose nature, suchas hardwired devices, field programmable gate arrays (FPGAs), digitalsignal processors (DSPs), or application specific integrated circuits(ASICs), can also be used without departing from the scope and spirit ofthe inventive concepts disclosed herein.

Where a method comprising a series of process steps is implemented by acomputer or a machine with use with the organ mimic device describedbelow and those process steps can be stored as a series of instructionsreadable by the machine, they can be stored on a tangible medium such asa computer memory device (e.g., ROM (Read Only Memory), PROM(Programmable Read Only Memory), EEPROM (Electrically ErasableProgrammable Read Only Memory), FLASH Memory, Jump Drive, and the like),magnetic storage medium (e.g., tape, magnetic disk drive, and the like),optical storage medium (e.g., CD-ROM, DVD-ROM, paper card, paper tapeand the like) and other types of program memory.

Embodiments of the present device can be applied in numerous fieldsincluding basic biological science, life science research, drugdiscovery and development, drug safety testing, chemical and biologicalassays, as well as tissue and organ engineering. In an embodiment, theorgan mimic device can be used as microvascular network structures forbasic research in cardiovascular, cancer, and organ-specific diseasebiology. Furthermore, one or more embodiments of the device findapplication in organ assist devices for liver, kidney, lung, intestine,bone marrow, and other organs and tissues, as well as in organreplacement structures.

The cellular responses to the various environmental cues can bemonitored using various systems that can be combined with the presentdevice. One can monitor changes in pH using well known sensors. One canintegrate force sensors into the membrane to measure changes in themechanical properties of the cells. One can also sample cells,continuously or periodically for measurement of changes in genetranscription or changes in cellular biochemistry or structuralorganization. For example, one can measure reactive oxygen species(ROSs) that are a sign of cellular stress. One can also subject the“tissue” grown on the membrane to microscopic analysis,immunohistochemical analysis, in situ hybridization analysis, or typicalpathological analysis using staining, such as hematoxylin and eosinstaining. Samples for these analysis can be carried out in real-time, ortaken after an experiment or by taking small biopsies at differentstages during a study or an experiment.

One can subject the cells grown on the membrane to other cells, such asimmune system cells or bacterial cells, to antibodies orantibody-directed cells, for example to target specific cellularreceptors. One can expose the cells to viruses or other particles. Toassist in detection of movement of externally supplied substances, suchas cells, viruses, particles or proteins, one can naturally label themusing typical means such as radioactive or fluorescent labels.

Cells can be grown, differentiated, cultured, supported or sustained,and/or analyzed using the present device for at least about 1 week, atleast about 2 weeks, at least about 3 weeks, at least about 4 weeks, atleast about 5 weeks, at least about 6 weeks, at least about 7 weeks, atleast about 8 weeks or longer. For example, as discussed below, it hasbeen shown that the cells can be maintained viable and differentiated ona membrane in an embodiment of the described device for at least about 1month or longer. In some embodiments, cells can be cultured in thedevice to induce cell growth. In some embodiments, cells (e.g., someprimary cells) can be sustained, rather than continue proliferating, inthe device.

The organ mimic device described herein has many different applicationsincluding, but not limited to, cell differentiation, formation of astratified and/or three-dimensional tissue structure, development of adisease model in a tissue of interest, development of a mucosal immunityplatform; studies on ciliary clearance of a particle; studies onairborne transmissibility of pathogens; studies on immune cell response(e.g., trans-epithelial migration, maturation, activation, cell killing,and/or drainage); studies on various tissue-specific diseases such asrespiratory, intestinal, digestive, skin, cardiac, and/or oculardiseases; studies of mechanism of action of drugs, target identificationand/or validation, identification of markers of disease; assessingpharmacokinetics and/or pharmacodynamics of various chemical orbiological agents; assessing efficacy of therapeutics and/or vaccines;testing gene therapy vectors; drug and/or vaccine development; moleculeor drug screening or drug discovery; determination of an appropriatetreatment or drug for a specific patient population or individualpatient; identification of a risk population to a disease or disorder;identification of a new drug target for a patient population that isnon-responsive to a previously-administered treatment; studies of cellbehavior in a physiologically-relevant model (including, e.g., stemcells and bone marrow cells); studies on biotransformation, absorption,clearance, metabolism, and activation of xenobiotics; studies onbioavailability and transport of chemical or biological agents acrossepithelial or endothelial layers; studies on transport of biological orchemical agents across the blood-brain barrier; studies on transport ofbiological or chemical agents across the intestinal epithelial barrier;studies on acute basal toxicity of chemical agents; studies on acutelocal or acute organ-specific toxicity of chemical agents; studies onchronic basal toxicity of chemical agents; studies on chronic local orchronic organ-specific toxicity of chemical agents; studies onteratogenicity of chemical agents; studies on genotoxicity,carcinogenicity, and/or mutagenicity of chemical agents; detection ofinfectious biological agents and/or biological weapons; detection ofharmful chemical agents and chemical weapons; studies on infectiousdiseases (e.g., bacterial, viral and/or fungal infections); assessinginfectivity and/or virulence of a new strain; studies on the optimaldose range of a chemical and/or biological agent to treat a disease;prediction of the response of an organ in vivo exposed to a biologicaland/or chemical agent; studies concerning the impact of genetic contenton response to agents; studies on gene transcription in response tochemical or biological agents; studies on protein expression in responseto chemical or biological agents; studies on changes in metabolism inresponse to chemical or biological agents; as well as example usesdescribed below. The organ mimic device can also be used to screen onthe cells, for an effect of the cells on the materials (for example, ina manner equivalent to tissue metabolism of a drug).

In some embodiments, the present device can be used to simulate themechanical load environment of walking, running, breathing, peristalsis,flow of flow or urine, or the beat of a heart, to cells cultured frommechanically active tissues, such as heart, lung, skeletal muscle, bone,ligament, tendon, cartilage, smooth muscle cells, intestine, kidney,endothelial cells and cells from other tissues. Rather than test thebiological or biochemical responses of a cell in a static environment,the investigator can apply a range of frequencies, amplitudes andduration of mechanical stresses, including tension, compression andshear, to cultured cells. For example, one can mechanically modulate themembrane within the device to simulate the mechanical load environmentof walking, running, breathing/respiration, and peristalsis.

A skilled artisan can place various types of cells on the surface(s) ofthe membrane. Cells include any cell type from a multicellularstructure, including nematodes, amoebas, up to mammals such as humans.Cell types implanted on the device depend on the type of organ or organfunction one wishes to mimic, and the tissues that comprise thoseorgans. More details of the various types of cells implantable on themembrane of the devices described herein are discussed below.

One can also co-culture various stem cells, such as bone marrow cells,induced adult stem cells, embryonic stem cells, induced pluripotent stemcells, or stem cells isolated from adult tissues on either one or bothsides of the membrane. Using different culture media in the chambersfeeding each layer of cells, one can allow different differentiationcues to reach the stem cell layers thereby differentiating the cells todifferent cell types. One can also mix cell types on the same side ofthe membrane to create co-cultures of different cells without membraneseparation.

Exemplary Microfluidic Devices and Methods of Uses Thereof

In general, the present disclosure is directed to device and method ofuse in which the device includes a body having a central channelseparated by one or more membranes. The membrane(s) are configured todivide the central channel into two or more closely apposed parallelchannels of substantially different heights, wherein one or more firstfluids are applied through at least one mesochannel and one or moresecond fluids are applied through at least one microchannel. The heightratio of the mesochannel(s) to the microchannel(s) is greater than 1:1.The surfaces of each membrane can be treated or coated with celladhesion molecules to support the attachment of cells and promote theirorganization into tissues on the upper and lower surface of themembrane, thereby creating a tissue-tissue interface separated by amembrane between the adjacent parallel fluid channels. The membrane canbe porous (e.g., permeable or selectively permeable), non-porous (e.g.,non-permeable), rigid, flexible, elastic, or any combination thereof. Insome embodiments, the membrane can be porous, e.g., allowingexchange/transport of fluids (e.g., gas and/or liquids), passage ofmolecules such as nutrients, cytokines and/or chemokines, celltransmigration, or any combinations thereof. In some embodiments, themembrane can be non-porous. Fluid pressure, flow and channel geometrycan be varied to apply a desired fluid shear stress to one or both cellor tissue layers. The larger mesochannel(s) provides a lower shear, morespacious environment for the cell or tissue layer cultured therein, ascompared to the cell or tissue layer cultured in the smallermicrochannel(s).

In a non-limiting example embodiment, the device can be configured tomimic operation of an airway or a bronchus, whereby cells that preferlower shear and/or a stratified structure, e.g., airway epithelialcells, are present on one surface of the membrane facing themesochannel, while lung capillary endothelial cells, fibroblasts, smoothmuscle cells are present on the opposite face of the same membranefacing the microchannel. The device thereby allows simulation of thestructure and function of a functional airway or bronchus unit that canbe exposed to physiological mechanical strain to simulate breathing orto both air-borne and blood-borne chemical, molecular, particulate andcellular stimuli to investigate the exchange of chemicals, molecules,and cells across this tissue-tissue interface through the pores of themembrane. The device impacts the development of in vitro airway orbronchus models that mimic organ-level responses which are able to beanalyzed under physiological and pathological conditions. This systemcan be used in several applications including, but not limited to,disease models, drug screening, drug delivery, vaccine delivery,biodetection, toxicology, physiology and organ/tissue engineeringapplications.

In other embodiments, the device can be adapted for other organ mimeticdevices requiring taller channel height to support optimal cell cultureincluding, but not limited to, skin-on-a-chip, heart-on-a-chip,liver-on-a-chip, gut-on-a-chip, and eye-on-a-chip. For example, theorgan mimetic devices described in the International Patent ApplicationNos. PCT/US12/68725 and PCT/US12/68766, the content of which areincorporated herein by reference, can be modified to have one of themicrochannels with a taller channel height.

FIG. 1 illustrates a block diagram of the overall system employing theinventive device in accordance with an embodiment. As shown in FIG. 1,the system 100 includes an organ mimic device 102, one or more fluidsources 104, 104 _(N) coupled to the device 102, one or more optionalpumps 106 coupled to the fluid source 104 and device 102. One or morecentral processing units (CPUs) 110 can be coupled to the pump 106 andpreferably control the flow of fluid in and out of the device 102. TheCPU 110 preferably includes one or processors 112 and one or morelocal/remote storage memories 114. A display 116 can be coupled to theCPU 110, and one or more pressure sources 118 can be coupled to the CPU110 and the device 102. The CPU 110 preferably controls the flow andrate of pressurized fluid to the device. It should be noted thatalthough one interface device 102 is shown and described herein, aplurality of interface devices 102 can be tested and analyzed within thesystem 100 as discussed below.

As will be discussed in more detail, the organ mimic device 102preferably includes two or more ports which place the mesochannels andmicrochannels of the device 102 in communication with the externalcomponents of the system, such as the fluid and pressure sources. Inparticular, the device 102 can be coupled to the one or more fluidsources 104 _(N) in which the fluid source can contain air, culturemedium, blood, water, cells, compounds, particulates, and/or any othermedia which are to be delivered to the device 102. In one embodiment,the fluid source 104 provides fluid to one or more mesochannels andmicrochannels of the device 102 and also preferably receives the fluidwhich exits the device 102. In some embodiments, the fluid exiting thedevice 102 can additionally or alternatively be collected in a fluidcollector or reservoir 108 separate from the fluid source 104. Thus, itis possible that separate fluid sources 104, 104 _(N) respectivelyprovide fluid to and remove fluid from the device 102.

In an embodiment, fluid exiting the device 102 can be reused andreintroduced into the same or different input port through which itpreviously entered. For example, the device 102 can be set up such thatfluid passed through a particular central sub-channel (e.g., mesochannelor microchannel) is recirculated back to the device and is again runthrough the same channel. This could be used, for instance, to increasethe concentration of an analyte in the fluid as it is recirculated thedevice. In another example, the device 102 can be set up such that fluidpassed through the device and is recirculated back into the device andthen subsequently run through another channel (e.g., mesochannel ormicrochannel). This could be used to change the concentration or makeupof the fluid as it is circulated through another channel (e.g.,mesochannel or microchannel).

One or more pumps 106 are preferably utilized to pump the fluid into thedevice 102, although pumps in general are optional to the system. Fluidpumps are well known in the art and are not discussed in detail herein.As will be discussed in more detail below, each microchannel portion ispreferably in communication with its respective inlet and/or outletport, whereby each microchannel portion of allow fluid to flowtherethrough.

Each mesochannel and microchannel in the device preferably has dedicatedinlet and outlet ports which are connected to respective dedicated fluidsources and/or fluid collectors to allow the flow rates, flow contents,pressures, temperatures and other characteristics of the media to beindependently controlled through each channel. Thus, one can alsomonitor the effects of various stimuli to each of the cell or tissuelayers separately by sampling the separate fluid channels for thedesired cellular marker, such as changes in gene expression at RNA orprotein level.

The cell injector/remover 108 component is shown in communication withthe device 102, whereby the injector/remover 108 is configured toinject, remove and/or manipulate cells, such as but not limited toepithelial, endothelial cells, fibroblasts, smooth muscle cells, basalcells, ciliated cells, columnar cells, goblet cells, muscle cells,immune cells, neural cells, hematopoietic cells, lung cells (e.g.,alveolar epithelial cells, airway cells, bronchial cells, trachealcells, and nasal epithelial cells), gut cells, brain cells, stem cells,skin cells, liver cells, heart cells, spleen cells, kidney cells,pancreatic cells, reproductive cells, and any combinations thereof, onone or more surfaces of the interface membrane within the device 102independent of cells introduced into the device via the inlet port(s)210, 218. For example, blood containing magnetic particles which pullpathogenic cells can be cultured in a separate device whereby themixture can be later introduced into the system via the injector at adesired time without having to run the mixture through the fluid source104. In an embodiment, the cell injector/remover 108 is independentlycontrolled, although the injector/remover 108 can be controlled by theCPU 110 as shown in FIG. 1. The cell injector/remover 108 is an optionalcomponent and is not necessary.

Although not required, the membrane of the device 102 can be adapted,e.g., by pneumatic means, to cause mechanical movements within thedevice 102. In these embodiments, an external force (e.g., mechanicalforce or pressure) can be applied from the one or more external forcesources 118 to cause mechanical movements of the membrane within thedevice 102. In an embodiment in which mechanical energy is used with thedevice, the external force source (e.g., stretching) 118 is controlledby the CPU 110 to stretch or release one or more membranes within thedevice to stretch and/or retract in response to the applied force. In anembodiment in which pressures are used with the device, the externalforce source (e.g., pressure source) 118 is controlled by the CPU 110 toapply a pressure differential within the device to effectively cause oneor more membranes within the device to stretch and/or retract inresponse to the applied pressure differential. In an embodiment, thepressure applied to the device 102 by the external force source (e.g.,pressure source) 118 is a positive pressure, depending on theconfiguration or application of the device. Additionally oralternatively, the pressure applied by the external force source (e.g.,pressure source) 118 is a negative pressure, such as vacuum or suction,depending on the configuration or application of the device. Theexternal force source 118 is preferably controlled by the CPU 110 toapply an external force (e.g., mechanical force or pressure) at settimed intervals or frequencies to the device 102, whereby the timingintervals can be set to be uniform or non-uniform. The external forcesource 118 can be controlled to apply uniform force (e.g., mechanicalforce or pressure) in the timing intervals or can apply different force(e.g., mechanical forces or pressures) at different intervals. Forinstance, the pressure applied by the pressure source 118 can have alarge magnitude and/or be set at a desired frequency to mimic a personrunning or undergoing exertion. The external force source 118 can alsoapply slow and/or irregular patterns, such as simulating a personsleeping or having a respiratory problem. In an embodiment, the CPU 110operates the external force source 118 to randomly vary intervals ofapplying an external force (e.g., mechanical force or pressure) to causecyclic stretching patterns to simulate irregularity in breath rate andtidal volumes during natural breathing.

In some embodiments, a gas-flow source generator 122 can be coupled tothe device 102 to introduce a gas inflow (e.g., an air inflow) to atleast one channel of the device (e.g., the mesochannel of the device tomimic respiration).

One or more sensors 120 can be coupled to the device 102 to monitor oneor more areas within the device 102, whereby the sensors 120 providemonitoring data to the CPU 110. One type of sensor 120 is preferably aforce sensor which provides data regarding the amount of force, stress,and/or strain applied to a membrane or pressure in one or more operatingchannels within the device 102. In one embodiment in which pressure isused within the device, pressure data from opposing sides of the channelwalls can be used to calculate real-time pressure differentialinformation between the operating and central sub-channels (e.g.,mesochannels and microchannels). The monitoring data would be used bythe CPU 110 to provide information on the device's operationalconditions as well as how the cells are behaving within the device 102in particular environments in real time. The sensor 120 can be anelectrode, have infrared, optical (e.g. camera, LED), or magneticcapabilities or utilize any other appropriate type of technology toprovide the monitoring data. For instance, the sensor can be one or moremicroelectrodes which analyze electrical characteristics across themembrane (e.g. potential difference, resistance, and short circuitcurrent) to confirm the formation of an organized barrier, as well asits fluid/ion transport function across the membrane. It should be notedthat the sensor 120 can be external to the device 102 or be integratedwithin the device 102. In some embodiments, the CPU 110 controlsoperation of the sensor 120, although it is not necessary. The data ispreferably shown on the display 116.

FIG. 2A illustrates a perspective view of the microfluidic device inaccordance with an embodiment. In particular, as shown in FIG. 2A, thedevice 200 (also referred to reference numeral 102) preferably includesa body 202 having a branched microchannel design 203 in accordance withan embodiment. The body 202 can be made of an elastomeric material,although the body can be alternatively made of a non-elastomericmaterial, or a combination of elastomeric and non-elastomeric materials.It should be noted that the microchannel design 203 is only exemplaryand not limited to the configuration shown in FIG. 2A.

The body 202 can be fabricated from a rigid material, an elastomericmaterial, or a combination thereof. As used herein, the term “rigid”refers to a material that is stiff and does not bend easily, ormaintains very close to its original form after pressure has beenapplied to it. The term “elastomeric” as used herein refers to amaterial or a composite material that is not rigid as defined herein. Anelastomeric material is generally moldable and curable, and has anelastic property that enables the material to at least partially deform(e.g., stretching, expanding, contracting, retracting, compressing,twisting, and/or bending) when subjected to a mechanical force orpressure and partially or completely resume its original form orposition in the absence of the mechanical force or pressure. In someembodiments, the term “elastomeric” can also refer to a material that isflexible/stretchable but does not resume its original form or positionafter pressure has been applied to it and removed thereafter. The terms“elastomeric” and “flexible” are interchangeably used herein.

In some embodiments, the material used to make the body 202 or at leastthe portion of the body 202 that is in contact with a gaseous and/orliquid fluid is preferably made of a biocompatible polymer or polymerblend, including but not limited to, polydimethylsiloxane (PDMS),polyurethane, polyimide, styrene-ethylene-butylene-styrene (SEBS),polypropylene, or any combinations thereof. As used herein, the term“biocompatible” refers to any material that does not deteriorateappreciably and does not induce a significant immune response ordeleterious tissue reaction, e.g., toxic reaction or significantirritation, over time when implanted into or placed adjacent to thebiological tissue of a subject, or induce blood clotting or coagulationwhen it comes in contact with blood.

In some embodiments, the body 202 can comprise an elastomeric portionfabricated from a styrenic block copolymer-comprising composition, e.g.,as described in the U.S. Provisional Application No. 61/919,181 filedDec. 20, 2013 and the corresponding PCT International Application No.PCT/US2014/071611, entitled “Organomimetic devices and methods of useand manufacturing thereof” filed concurrently with the currentapplication on Dec. 19, 2014, can be adopted in the devices describedherein, the content of which is incorporated herein by reference. Insome embodiments, the styrenic block copolymer-comprising compositioncan comprise SEBS and polypropylene.

Additionally or alternatively, at least a portion of the body 202 can bemade of non-flexible or rigid materials like glass, silicon, hardplastic, metal, or any combinations thereof.

The membrane 208 can be made of the same material as the body 202 or amaterial that is different from the body 202 of the device. In someembodiments, the membrane 208 can be made of a rigid material. In someembodiments, the membrane is a thermoplastic rigid material. Examples ofrigid materials that can be used for fabrication of the membraneinclude, but are not limited to, polyester, polycarbonate or acombination thereof. In some embodiments, the membrane 208 can comprisea flexible material, e.g., but not limited to PDMS.

In some embodiments, the body of the device and/or the membrane cancomprise or is composed of an extracellular matrix polymer, gel, and/orscaffold. Any extracellular matrix can be used herein, including, butnot limited to, silk, chitosan, elastin, collagen, proteoglycans,hyaluronic acid, collagen, fibrin, and any combinations thereof.

The device in FIG. 2A includes a plurality of access ports 205 whichwill be described in more detail below. In addition, the branchedconfiguration 203 includes a tissue-tissue interface simulation region(membrane 208 in FIG. 2B) where cell behavior and/or passage of gases,chemicals, molecules, particulates and cells are monitored. FIG. 2Billustrates an exploded view of the organ mimic device in accordancewith an embodiment. In particular, the outer body 202 of the device 200is preferably comprised of a first outer body portion 204, a secondouter body portion 206 and an intermediary membrane 208 configured to bemounted between the first and second outer body portions 204, 206 whenthe portions 204, 206 are mounted to one another to form the overallbody.

The first outer body portion 204 can have a thickness of any dimension,depending, in part, on the height of the mesochannel 250A. In someembodiments, the thickness of the first outer body portion 204 can beabout 1 mm to about 100 mm, or about 2 mm to about 75 mm, or about 3 mmto about 50 mm, or about 3 mm to about 25 mm. In one embodiment, thethickness of the first outer body portion 204 can be about 4.8 mm. Insome embodiments, the first outer body portion 204 can have a thicknessthat is more than the height of the mesochannel by no more than 500microns, no more than 400 microns, no more than 300 microns, no morethan 200 microns, no more than 100 microns, no more than 70 microns orless. In some embodiments, it is desirable to keep the first outer bodyportion 204 as thin as possible such that cells on the membrane can bevisualized or detected by microscopic, spectroscopic, and/or electricalsensing methods.

The second outer body portion 206 can have a thickness of any dimension,depending, in part, on the height of the microchannel 250B. In someembodiments, the thickness of the second outer body portion 206 can beabout 50 μm to about 10 mm, or about 75 μm to about 8 mm, or about 100μm to about 5 mm, or about 200 μm to about 2.5 mm. In one embodiment,the thickness of the second outer body portion 206 can be about 1 mm toabout 1.5 mm. In one embodiment, the thickness of the second outer bodyportion 206 can be about 0.2 mm to about 0.5 mm. In some embodiments,the second outer body portion 206 can have a thickness that is more thanthe height of the microchannel by no more than 500 microns, no more than400 microns, no more than 300 microns, no more than 200 microns, no morethan 100 microns, no more than 70 microns or less. In some embodiments,it is desirable to keep the second outer body portion 206 as thin aspossible such that cells on the membrane can be visualized or detectedby microscopic, spectroscopic, and/or electrical sensing methods.

FIG. 2B illustrates an exploded view of the device in accordance with anembodiment. As shown in FIG. 2B, the first outer body portion 204includes one or more inlet fluid ports 210 in communication with one ormore corresponding inlet apertures 211 located on an outer surface ofthe body 202. The device 100 is preferably connected to the fluid source104 via the inlet aperture 211 in which fluid travels from the fluidsource 104 into the device 100 through the inlet fluid port 210.

Additionally, the first outer body portion 204 includes one or moreoutlet fluid ports 212 in communication with one or more correspondingoutlet apertures 215 on the outer surface of the body 202. Inparticular, fluid passing through the device 100 exits the device 100 toa fluid collector 108 or other appropriate component via thecorresponding outlet aperture 215. It should be noted that the device200 can be set up such that the fluid port 210 is an outlet and fluidport 212 is an inlet.

In some embodiments, as shown in FIGS. 2B and 211, the device 200 cancomprise an inlet channel 225 connecting an inlet fluid port 210 to thecentral channel 230. The inlet channels and inlet ports can be used tointroduce cells, agents (e.g., but not limited to, stimulants, drugcandidate, particulates), air flow, and/or cell culture media into themesochannel 250A and microchannel 250B.

The central and operating channels described herein are shown generallyas linear. It is to be understood, however, that the channels can besubstantially linear or they can be non-linear. Thus, the presentinventive concepts are not limited to straight or linear channels andcan comprise curved, angled, or otherwise non-linear channels, e.g.,central channel and/or operating channel(s). It is to be furtherunderstood that a first portion of a channel (e.g., central channeland/or operating channel(s)) can be straight, and a second portion ofthe same channel can be curved, angled, or otherwise non-linear.Generally, the non-linear channel comprises at least one (e.g., one,two, three, four, five, six, seven, eight, nine, ten or more) curved orangled sections. A non-linear channel can also comprise at least one(e.g., one, two, three, four, five, six, seven, eight, nine, ten ormore) substantially linear sections.

Generally, non-linear section has curve or angle in the range from about5° to about 175°. In some embodiments, the non-linear section comprisesa curve or angle of about 80° to about 100°. In some embodiments, anon-linear section joins two substantially linear sections that aresubstantially parallel to each other. In some embodiments, a non-linearsection joins two substantially linear sections that are substantiallyperpendicular to each other. In some embodiments, a non-linear sectionjoins two substantially linear sections that are positioned at an angleless than perpendicular to each other. In some embodiments, a non-linearsection joins two substantially linear sections that are positioned atan angle higher than perpendicular to each other.

FIG. 23 illustrates an embodiment of device 200 that comprises anon-linear central channel (230) and operating channels 252. The centralchannel 200 comprises one or more non-linear sections (280) that jointogether two linear sections (290) of the central channel. Withoutwishing to be bound by a theory, a non-linear central channel canincrease the ratio of culture area to chip area, thereby providing alarger surface area for cells to grow. This can also allow for a higheramount or density of cells in the central channel.

In some embodiments, the device comprises a non-linear central channel,wherein height of the mesochannel is about 1 mm and height of themicrochannel is about 200 μm. In some further embodiments of thisdevice, the height of the operating channels is about 500 μm. In someembodiments, the height of the operating channels can be greater thanthe height of the mesochannel or microchannel, or the combined height ofthe mesochannel and the microchannel.

The device 200 can also comprise an outlet channel 227 connecting anoutlet fluid port 212 to the central channel 230. The outlet channelsand outlet ports can also be used to introduce cells, agents (e.g., butnot limited to, stimulants, drug candidate, particulates), air flow,and/or cell culture media into the mesochannel 250A and microchannel250B.

Although the inlet and outlet apertures 211, 215 are shown on the topsurface of the body 202 and are located perpendicular to the inlet andoutlet channels 225, 227, one or more of the apertures 211, 215 can belocated on one or more lateral surfaces of the body such that at leastone of the inlet and outlet apertures 211, 215 can be in-plane with theinlet and/or outlet channels 225, 227, respectively, and/or be orientedat an angle from the plane of the inlet and/or outlet channels 225, 227.For example, FIG. 2C shows a perspective view of the device with theinlet and outlet apertures configured on the lateral surfaces of thebody in accordance with one embodiment. By placing the inlet and outletapertures on the lateral surfaces of the body, the inlet channels 231and outlet channels 233 can form an angle of less than 90 degrees (e.g.,ranging between 10 degrees and 50 degrees) with the mesochannel 250Aand/or microchannel 250B. In one embodiment, the inlet channels 231 andoutlet channels 233 can form an angle of about 25 degrees with themesochannel 250A and/or microchannel 250B. In one embodiment, the inletchannels 231 and outlet channels 233 can form an angle of about 45degrees with the mesochannel 250A and/or microchannel 250B. These angledconfigurations can reduce or prevent accumulation of cells upon cellseeding process at the ports and/or formation of cell plugs. Inaddition, the design of the inlet and outlet apertures 211, 215 on thelateral surfaces of the body can allow access to both the mesochanneland microchannel, which can be used, e.g., to remove bubbles withmicroinjection tips in the bottom channel, access the cells, wound thecells, and/or inject new cell type into the device.

In an embodiment, the inlet fluid port 210 and the outlet fluid port 212are in communication with the mesochannel 250A (see FIG. 2D) such thatfluid can dynamically travel from the inlet fluid port 210 to the outletfluid port 212 via the mesochannel 250A, independently of themicrochannel 250B (see FIG. 2D).

In another embodiment, the fluid passing between the inlet and outletfluid ports can be shared between the mesochannel 250A and microchannel250B. In either embodiment, characteristics of the fluid flow, such asflow rate, fluid type and/or composition, and the like, passing throughthe mesochannel 250A can be controllable independently of fluid flowcharacteristics through the microchannel 250B and vice versa.

In one embodiment, the first portion 204 includes one or more pressureinlet ports 214 and one or more pressure outlet ports 216 in which theinlet ports 214 are in communication with corresponding apertures 217located on the outer surface of the device 100. Although the inlet andoutlet apertures are shown on the top surface of the body 202, one ormore of the apertures can alternatively be located on one or morelateral sides of the body. In operation, one or more pressure tubes (notshown) connected to the external force source (e.g., pressure source)118 (FIG. 1) provides positive or negative pressure to the device viathe apertures 217. Additionally, pressure tubes (not shown) areconnected to the device 100 to remove the pressurized fluid from theoutlet port 216 via the apertures 223. It should be noted that thedevice 200 can be set up such that the pressure port 214 is an outletand pressure port 216 is an inlet. It should be noted that although thepressure apertures 217, 223 are shown on the top surface of the body202, one or more of the pressure apertures 217, 223 can be located onone or more side surfaces of the body 202.

Referring to FIG. 2B, the second portion 206 includes one or more inletfluid ports 218 and one or more outlet fluid ports 220. As shown in FIG.2B, the inlet fluid port 218 is in communication with aperture 219 andoutlet fluid port 220 is in communication with aperture 221, whereby theapertures 219 and 221 are located on the outer surface of the secondouter body portion 206. Although the inlet and outlet apertures areshown on the surface of the body 202, one or more of the apertures canbe alternatively located on one or more lateral sides of the body, e.g.,as shown in FIG. 2C.

As with the first outer body portion 204 described above, one or morefluid tubes connected to the fluid source 104 (FIG. 1) are preferablycoupled to the aperture 219 to provide fluid to the device 100 via port218. Additionally, fluid exits the device 100 via the outlet port 220and out aperture 221 to a fluid reservoir/collector 108 or othercomponent. It should be noted that the device 200 can be set up suchthat the fluid port 218 is an outlet and fluid port 220 is an inlet.

In one embodiment, the second outer body portion 206 includes one ormore pressure inlet ports 222 and one or more pressure outlet ports 224.In particular, it is preferred that the pressure inlet ports 222 are incommunication with apertures 227 and pressure outlet ports 224 are incommunication with apertures 229, whereby apertures 227 and 229 arelocated on the outer surface of the second portion 206. Although theinlet and outlet apertures are shown on the bottom surface of the body202, one or more of the apertures can be alternatively located on one ormore lateral sides of the body. Pressure tubes connected to the externalforce source (e.g., pressure source) 118 (FIG. 1) can be engaged withports 222 and 224 via corresponding apertures 227 and 229. It should benoted that the device 200 can be set up such that the pressure port 222is an outlet and fluid port 224 is an inlet.

In some embodiments where the operating channels as described below(e.g., 252 shown in FIG. 2D) are not mandatory, the first portion 204does not require any pressure inlet port 214, pressure outlet port 216.Similarly, the second portion 206 does not require any pressure inletport 222 or pressure outlet port 224.

In an embodiment, the membrane 208 is mounted between the first portion204 and the second portion 206, whereby the membrane 208 is locatedwithin the body 202 of the device 200 (see FIG. 2D). In an embodiment,the membrane 208 is a made of a material having a plurality of pores orapertures therethrough, whereby molecules, cells, fluid or any media iscapable of passing through the membrane 208 via one or more pores in themembrane 208. As discussed in more detail below, the membrane 208 in oneembodiment can be made of a material which allows the membrane 208 toundergo stress and/or strain in response to an external force (e.g.,cyclic stretching or pressure). In one embodiment, the membrane 208 canbe made of a material which allows the membrane 208 to undergo stressand/or strain in response to pressure differentials present between themesochannel 250A, the microchannel 250B and the operating channels 252.Alternatively, the membrane 208 is relatively inelastic or rigid inwhich the membrane 208 undergoes minimal or no movement while media ispassed through one or more of the central sub-channels 250A, 250B and/orcells organize and move between the central sub-channels 250A, 250B viathe membrane.

Referring FIG. 2E illustrates a perspective view of the tissue-tissueinterface region of the first outer portion 204 of the body taken atline C-C (from FIG. 2B). As shown in FIG. 2E, the top portion of thetissue-tissue interface region 207A is within the body of the firstportion 204 and includes a top portion of a central channel 230(mesochannel 250A) and one or more top portion side operating channels252A located adjacent to the mesochannel 250A. Channel walls 234, 244preferably separate the central channel 230 from the operating channels252 such that fluid traveling through the central channel 230 does notpass into operating channels 252. Likewise, the channel walls 234, 244prevent pressurized fluid passing along operating channels 252 fromentering the mesochannel 250A. It should be noted that a pair ofoperating channels 252 are shown on opposing sides of central channel230 in FIG. 2D, however the device can incorporate more than twooperating channels 252. In some embodiments that the device 200 caninclude only one operating channel 252 adjacent to the central channel230.

FIG. 2F illustrates a perspective view of the tissue interface regiontaken at line D-D of the second outer portion 206 of the body. As shownin FIG. 2F, the tissue interface region includes a bottom portion of thecentral channel 230 (microchannel 250B) and at least two bottom portionsof operating channels 252B located adjacent to the microchannel 250B. Apair of channel walls 234, 244 preferably separate the central channel230 from the operating channels 252 such that fluid traveling throughthe central channel 230 does not pass into operating channels 232.Likewise, the channel walls 234, 244 prevent pressurized fluid passingalong operating channels 232 from entering the central channel 230.

The central channel 230 can have a length suited to the need of anapplication (e.g., a physiological system to be modeled), desirable sizeof the device, and/or desirable size of the view of field. In someembodiments, the central channel 230 can have a length of about 0.5 cmto about 10 cm. In one embodiment, the central channel 230 can have alength of about 1 cm to about 2 cm.

As shown in FIGS. 2E and 2F, the top and bottom portions of the centralchannel 230 each have a range of width dimension (shown as B) between200 microns and 10 mm, or between 200 microns and 1500 microns, orbetween 400 microns and 1000 microns, or between 50 and 2000 microns. Insome embodiments, the dimensions of the devices described herein can beconfigured to provide a low shear stress on epithelial cells whilesubmerged in liquid culture (which can be subsequently subjected to anair-liquid interface (ALI) induction). For example, in some embodiments,the width of the channels (mesochannels and microchannels) can be atleast or greater than 400 μm or more, including, e.g., at least orgreater than 500 μm, at least or greater than 600 μm, at least orgreater than 700 μm, at least or greater than 800 μm, at least orgreater than 900 μm, at least or greater than 950 μm or more. In oneembodiment, the top and bottom portions of the central channel (250Amesochannel and 250B microchannel) each have a width dimension ofgreater than 400 μm. In one embodiment, the top and bottom portions ofthe central channel (250A mesochannel and 250B microchannel) each have awidth dimension of about 1 mm. It should be noted that other widthdimensions (e.g., greater than 10 mm or smaller than 50 microns) can beused depending on the type of physiological system which is beingmimicked in the device, and/or the number ofmesochannel(s)/microchannel(s) formed in the central channel, which willbe discussed further below. Thus, in some embodiments, the width of thecentral channel can be between 400 microns and 50 mm, or between 400microns and 10 mm, or between 800 microns and 5 mm, or between 100microns and 10 mm.

In some embodiments where the top portion of the central channel 230forms a single mesochannel 250A, the width of the mesochannel 250A isessentially the same width of the central channel 230. Similarly, insome embodiments where the bottom portion of the central channel 230forms a single microchannel 250B, the width of the microchannel 250B isessentially the same width of the central channel 230.

In some embodiments where the top portion of the central channel 230forms at least two or more mesochannels, e.g., as shown in FIG. 22B, thewidth of the mesochannels 2250A and 2250B are smaller than the width ofthe central channel 230. In some embodiments where the bottom portion ofthe central channel 230 forms at least two or more microchannels, e.g.,as shown in FIG. 22A, the width of the microchannels 2260A and 2260B aresmaller than the width of the central channel 230. Multiple mesochannelsand/or microchannels formed in a central channel are further describedin detail below.

In some embodiments, the width of the two channels can be configured tobe different, with the centers of the channels aligned or not aligned.In some embodiments, the channel heights, widths, and/or cross sectionscan vary along the length the devices described herein.

In accordance with some embodiments described herein, the height of atleast a length portion of the mesochannel 250A (e.g., the length portionwhere the cells are desired to grow and form a stratified,pseudostratified or 3-dimensional tissue structure) is substantiallygreater than the height of the microchannel 250B within the same lengthportion. For example, the height ratio of the mesochannel to themicrochannel is greater than 1:1, including, for example, greater than1.1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1,25:1, 30:1, 35:1, 40:1, 45:1, 50:1. In some embodiments, the heightratio of the mesochannel to the microchannel can range from 1.1:1 toabout 50:1, or from about 2.5:1 to about 50:1, or from 2.5 to about25:1, or from about 5:1 to about 25:1. In one embodiment, the heightratio of the mesochannel to the microchannel ranges from about 10:1 toabout 20:1. The higher mesochannel can offer a reduced stressenvironment and increased overhead space for growth of cells thatrequire low shear and more space to form a stratified structure and/or athree-dimensional tissue.

In some embodiments, the height of at least a length portion of themesochannel 250A can be sufficient to accommodate the tallest cell(including any projections from the cell such as cilia) or the thickestcell present on the membrane facing the mesochannel.

In some embodiments, the height of at least a length portion of themesochannel 250A can have a dimension sufficient to permit growth ofmore than one cell layers, e.g., 2 cell layers, 3 cell layers, 4 celllayers, 5 cell layers, 6 cell layers, or more. The cell layers can eachbe functionally and/or morphologically the same or different. The heightof the mesochannel can vary with the thickness of at least a portion ofa biological tissue or organ to be modeled. For example, in someembodiments, the height of the mesochannel 250A can have a dimensionsufficient to form a stratified structure (a structure comprising cellsarranged in layers) of an airway epithelium comprising ciliated cellsand mucus-secreting cells, e.g., as shown in FIG. 5B. In someembodiments, the height of the mesochannel 250A can have a dimensionsufficient to form a stratified structure of a small airway epithelium.In some embodiments, the mesochannel 250A can have a height dimensionconfigured to permit formation of a skin equivalent to model the skin(e.g., a mammalian or animal skin) as an organ.

In some embodiments, the height of the mesochannel 250A can beconfigured to provide sufficient overhead space above astratified/pseudostratified or three-dimensional structure for an airflow such that air shear stress on the cells (e.g., airway or skinepithelial cells) can be maintained within a physiological range (e.g.,between 0.01 dynes/cm² and 1700 dynes/cm²). In one embodiment, the airflow can be maintained as a static flow.

In some embodiments, the height of the mesochannel 250A can have adimension sufficient for formation of a three-dimensional tissue. Forexample, the height of the mesochannel 250A can have a dimensionsufficient for formation of a three-dimensional gut or intestinaltissue, where the intestinal epithelial cells grow into folds thatrecapitulate the structure of intestinal villi. In some embodiments, theheight of the mesochannel 250A can be configured to provide sufficientoverhead space above the three dimensional structure for a liquid flowsuch that liquid shear stress on the cells (e.g., intestinal epithelialcells) can be maintained within a physiological range (e.g., between0.01 dynes/cm² and 1700 dynes/cm²).

In some embodiments, the height of the mesochannel 250A can depend onaspect ratio of the height of the mesochannel 250A to the width of thecentral channel 230. The aspect ratio of the height of the mesochannel250A to the width of the central channel 230 can range from about 1:5 toabout 50:1 or about 1:10 to about 20:1. In some embodiments, the heightof the mesochannel 250A can range from about 100 μm to about 50 mm,about 150 μm to about 25 mm, or about 200 μm to about 10 mm. In someembodiments, the height of the mesochannel 250A can range from about 100μm to about 5 mm, about 150 μm to about 2.5 mm, or about 200 μm to about2 mm. In one embodiment, the height of the mesochannel 250A is about 220μm to about 1 mm. In one embodiment, the height of the mesochannel 250Ais about 100 μm to about 5 mm. In one embodiment, for a 1 mm widechannel, the height of the mesochannel can range from about 100 μm toabout 20 mm.

The mesochannel can have a uniform height along the length of themesochannel. Alternatively, the mesochannel can have a varying heightalong the length of the mesochannel. For example, a length portion ofthe mesochannel (e.g., where a stratified/pseudo-stratified orthree-dimensional tissue structure is desired to be formed therein) canbe substantially taller than the same length portion of themicrochannel, while the rest of the mesochannel can have a heightcomparable to or even smaller than the height of the microchannel.

The height of the microchannel 250B can be of any dimension providedthat the flow rate and/or shear stress of a medium flowing in themicrochannel can be maintained within a physiological range, or does notcause any adverse effect to the cells, and/or there is sufficient spacefor the cell growth on the surface of the membrane facing themicrochannel. For example, in some embodiments, the height of themicrochannel 250B can be designed to mimic a blood vessel channel inwhich blood or cell culture medium flows at a physiological fluidpressure and/or flow rate.

Accordingly, in some embodiments, the height of the microchannel 250Bcan be substantially smaller than the height of the mesochannel 250A.For example, the height of the microchannel can be about 1% to about80%, or about 5% to about 70%, or about 10% to about 50%, of the heightof the mesochannel. In some embodiments, the height of the microchannelcan be no more than 30%, no more than 20%, no more than 10%, of theheight of the mesochannel. In some embodiments, the height of themicrochannel is no more than 10% of the height of the mesochannel.

In alternative embodiments, the height of the microchannel 250B can besubstantially the same as the height of the mesochannel 250A.

In some embodiments, the height of the microchannel 250B can range fromabout 1 μm to about 5 mm, about 10 μm to about 5 mm, about 25 μm to 2.5mm, or about 50 μm to about 1 mm. In some embodiments, the height of themicrochannel 250B can range from about 25 μm to about 1 mm, about 50 μmto about 750 μm, or about 75 μm to about 500 μm. In one embodiment, theheight of the microchannel 250B is about 50 μm to about 150 μm. In oneembodiment, the height of the microchannel 250B is about 100 μm to about160 μm.

In some embodiments, the body of the device can be further adapted toprovide mechanical modulation of the membrane within the centralchannel. Mechanical modulation of the membrane can include any movementof the membrane that is parallel to and/or perpendicular to theforce/pressure applied to the membrane, including, but are not limitedto, stretching, bending, compressing, vibrating, contracting, waving, orany combinations thereof. By way of example only, FIG. 2D illustrates asectioned view of the cell culture interface region within the body inaccordance with an embodiment where the membrane can be mechanicallymodulated by a pneumatic mechanism. In this embodiment where thepressure is applied within the device to mechanically modulate themembrane 208, the operating channel(s) 252 can be symmetrically arrangedaround the membrane 208. For example, the top half of the operatingchannel(s) 252A are formed in a bottom surface of the top body portion204 and the bottom half of the operating channel(s) 252B are formed in atop surface of the bottom body portion 206 such that when the two bodyportions 204 and 206 are mated to each other with a membrane 208positioned between the mesochannel 250A and the microchannel 250Bwhereby the side walls 228 and 238 as well as the channel walls 234, 244form the overall central channel 230 and operating channels 252, theplane 208P along the membrane 208 can bisect the operating channel(s)252 into the top half and bottom half of the operating channel(s) 252Aand 252B.

The width of the operating channels 252 can be of any dimension providedthat the aspect ratio of the height to width of the operating channels252 allows a sufficient mechanical force to be applied to the membraneand/or yields sufficient mechanical strength to withstand application ofcyclic pressures. In some embodiments, the width of the operatingchannels 252 can range from about 100 μm to about 5 mm or from about 200μm to about 2 mm.

In some embodiments, e.g., as shown in FIG. 2D, the heights of theoperating channels 252 can be smaller than the height of the centralchannel 230. In some embodiments, the heights of the operating channels252 can be no more than 70% of the height of the central channel 230,including, e.g., no more than 60%, no more than 50%, no more than 40%,no more than 30%, no more than 20% or less, of the height of the centralchannel 230. In some embodiments, the heights of the operating channels252 can be about 1.5 times to about 2.5 times the height of themicrochannel 250B. In some embodiments, the heights of the operatingchannels 252 can range from about 20 μm to about 10 mm, about 50 μm to 5mm, or about 100 μm to about 2 mm. In some embodiments, the height ofthe operating channels 252 can range from about 50 μm to about 2 mm,about 100 μm to about 1.5 mm, or about 150 μm to about 1000 μm. In oneembodiment, the height of the operating channels 252 is about 100 μm toabout 300 μm. In one embodiment, the height of the operating channels252 is about 200 μm to about 800 μm.

In some embodiments, e.g., as shown in FIGS. 24A-24B, the heights of theoperating channels 2452 can be greater than the height of the centralchannel 2430. In some embodiments, the heights of the operating channels2452 can be greater than the height of the central channel 2430 by atleast about 5%, including, e.g., at least about 10%, at least about 20%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, or more. In some embodiments, the heights of theoperating channels 2452 can be greater than the height of the centralchannel 2430 by at least about 1.1-fold, including, e.g., at least about1.5-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold or higher. In someembodiments, it can be desirable to have the heights of the operatingchannels larger than the height of the central channel. Without wishingto be bound by theory, increasing the heights of the operating channelsgenerally provides a larger surface area of the channel wall between theoperating channels and the central channel (e.g., 2434 and 2444 as shownin FIGS. 24A-24B), which can in turn allow a larger force acting on thechannel wall to flex in response to a pressure differential between theoperating channels and the central channel. In some embodiments, athicker channel wall between the operating channels and the centralchannel can be used, when a larger force is acted on the channel wall.

In some embodiments, the heights of the operating channels can besubstantially same as the height of the central channel.

Using FIGS. 2E and 2F as examples, in some embodiments, the top andbottom portions of the operating channels (252A and 252B) can each havea width dimension (shown as A) between 25 and 5000 microns, or between200 microns and 2000 microns, although other width dimensions can beused, e.g., depending on the amount of the mechanical force applied tothe membrane. While FIGS. 2E and 2F shows a uniform width dimension(shown as B) along at least a portion of the central channel height inthe device, the central channel can also have a non-uniform widthdimension B along at least a portion of its height in the device.

The channel walls 234, 244 can have any thickness that would permit thechannel walls to flex in response to a pressure differential between theoperating channels and the central channel 230. In some embodiments, thechannel walls 234, 244 can have a thickness range between 5 microns to500 microns, although other width dimensions can be used depending onthe material used for the walls, application in which the device isused. In one embodiment, the channel walls 234, 244 can have a thicknessrange between 50 microns to 500 microns or between 70 microns and 300microns.

The membrane 208 is oriented along a plane 208P parallel to the x-yplane within the central channel 230 shown in FIG. 2D. It should benoted that although one membrane 208 is shown in the central channel230, more than one membrane 208 can be configured within the centralchannel 230, as discussed in more detail below. In addition to beingpositioned within the central channel 250, the membrane 208 issandwiched in place by channel walls 234, 244 during formation of thedevice.

In some embodiments, the membrane 208 can separate the central channel250 into two or more distinct central sub-channels 250A and 250B, ofwhich at least one is a mesochannel 250A.

As will be discussed in further detail below, the membrane 208 can benon-porous or can have at least a portion which is sufficiently porousto allow cells and/or molecules to pass therethrough. The membrane 208can be rigid or flexible. In some embodiments, the membrane 208 is arigid porous membrane. In other embodiments, the membrane 208 is aflexible porous membrane. At least a portion of the membrane 208 canhave elastic or ductile properties which allow the membrane 208 to bemanipulated to stretch/retract along one or more planar axe. Thus, insome embodiments, one or more portions of the membrane 208 can be porousand elastic or porous, but inelastic. In some embodiments, the membrane208 can be non-porous and elastic or non-porous but inelastic.

A pressure differential can be applied within the device to causerelative continuous expansion and contraction of the membrane 208 alongthe x-y plane. In particular, as stated above, one or more pressuresources can apply pressurized fluid (e.g., air) along the one or moreoperating channels 252, whereby the pressurized fluid in the operatingchannels 252 creates a pressure differential on the channel walls 234,244.

In the embodiments shown in FIG. 2D, the pressurized fluid is a vacuumor suction force that is applied only through the operating channels252. The difference in pressure caused by the suction force against thechannel walls 234, 244 causes the walls 234, 244 to bend or bulgeoutward toward the sides of the device 228, 238 (see FIGS. 2E-2F).Considering that the membrane 208 is mounted to and sandwiched betweenthe channel walls 234, 244, the relative movement of the walls 234, 244thereby causes the opposing ends of the membrane to move along with thewalls to stretch along the membrane's plane. This stretching mimics themechanical forces experienced by a tissue-tissue interface, for example,in the airway or bronchus during breathing, and thus provides therelevant regulation for cellular self assembly into tissue structuresand cell behavior.

When the negative pressure is no longer applied (and/or positivepressure is applied to the operating channels), the pressuredifferential between the operating channels 252 and the central channel230 decreases and the channel walls 234, 244 retract toward theirneutral position or their original position prior to the application ofthe negative pressure. During operation, the negative pressure isalternately applied in timed intervals to the device 200 to causecontinuous expansion and contraction of the membrane 208 along itsplane, thereby simulating within a controlled in vitro environment aphysiological strain that is substantially the same as the stainproduced by motion associated with operation of the tissue-tissueinterface of the living organs, e.g., but not limited to breathing,peristalsis, or heart beating. As will be discussed, this mimicked organoperation within the controlled environment allows development ofdifferent corresponding organ-associated disease models. For example,the device described herein can be used to simulate breathing motion inan airway or bronchus and thus allows development of disease modelsassociated with breathing and airway constriction, such as asthma, andchronic obstructive pulmonary disease (COPD). In some embodiments, thedevice can be used to simulate other diseases models such as pulmonaryhypertension, radiation induced injury, cystic fibrosis or airbornediseases such as viral or bacterial infection, e.g., by culturingappropriate types of cells on at least one or both surfaces of themembrane 208 and inducing disease phenotypes in the cells, e.g., byusing physical, chemical and/or biological agents. Cell behavior can bemonitored within the device, as well as passage of molecules, chemicals,particulates and cells with respect to the membrane and the associatedfirst and second microchannels 250A, 250B.

It should be noted that the term pressure differential in the presentspecification relates to a difference of pressure on opposing sides of aparticular wall between the central channel and the outer operatingchannel. In some embodiments, the pressure differential can be createdin a number of ways to achieve the goal of expansion and/or contractionof the membrane 208. As stated above, a negative pressure (i.e. suctionor vacuum) can be applied to one or more of the operating channels 252.Alternatively, the membrane 208 can be pre-loaded or pre-stressed to bein a stretched state by default such that the channel walls 234, 244 arealready in the bent configuration. In this embodiment, positive pressureapplied to the operating channel 252 will create the pressuredifferential which causes the channel walls 234, 244 to move inwardtoward the central channel to contract the membrane 208.

In another embodiment, a combination of positive and negative pressureis applied to one or more operating channels 252 to cause movement ofthe membrane 208 along its plane in the central channel. In any of theabove embodiments, it is desired that the pressure of the fluid in theone or more operating channels 252 be such that a pressure differentialis in fact created with respect to the pressure of the fluid(s) in oneor more of the central channel(s) 250A, 250B to cause relativeexpansion/contraction of the membrane 208. For example, fluid having acertain pressure can be applied within the top central channel 250A,whereby fluid in the bottom central channel 250B can have a differentpressure. In this example, pressure applied to the one or more operatingchannels 252 must take into account the pressure of the fluid in eitheror both of the central channels 250A, 250B to ensure desiredexpansion/contraction of the membrane 208.

It is possible, in an embodiment, for a pressure differential to existbetween the top and bottom microchannels 250A, 250B to cause at least aportion of the membrane 208 to stretch and/or retract vertically in thez-direction in addition to expansion/contraction along the x-y plane.

In an embodiment, the expansion and retraction of the membrane 208 inturn applies mechanical forces to the adherent cells and ECM that mimicphysiological mechanical cues that can influence transport of chemicals,molecules particulates, and/or fluids or gas across the tissue-tissueinterface, and alter cell physiology. It should be noted that althoughmechanical modulation of the membrane created by pressure differentialsbetween the operating channels 252 and the central channel 230 is shownin FIG. 2D, in other embodiments, mechanical means, such as micromotorsor actuators, or any means that can cause the movement of the membrane,including use of one or more magnetic forces, can be employed to assistor substitute for the pressure differential to provide mechanicalmodulation of the membrane within the central channel, e.g., to modulatecell physiology. The membrane can be mechanically modulated to move inany direction, e.g., within the plane 208P and/or transverse to theplane 208P. In some embodiments, the membrane can be mechanicallymodulated to move along a single axis within or transverse to the plane208P. In alternative embodiments, the membrane can be mechanicallymodulated to move along at least two predefined axes, e.g., the axesthat define the plane 208P. Other example means of mechanical modulationof the membrane, e.g., as described in the U.S. Provisional ApplicationNo. 61/919,181 filed Dec. 20, 2013 and the corresponding PCTInternational Application No. PCT/US2014/071611, entitled “Organomimeticdevices and methods of use and manufacturing thereof” filed concurrentlywith the current application on Dec. 19, 2014, the content of which isincorporated herein by reference, can be adopted in the devicesdescribed herein.

In some embodiments, one or more of the channels can be configured tochange direction along the lengths of the channels, for example, usingcurved or sharp bends. This can provide a means to enable the directionof membrane modulation to vary along the length of the channel.

While FIGS. 2A-2F illustrate devices 200 comprising operating chambers252 (for mechanical modulation of the membrane 208), the device 200 doesnot require the operating channels 252 where the mechanical modulationof the membrane is not mandatory, e.g., where respiration through anairway is to be simulated. For example, as shown in FIG. 2G, the device201 comprises a mesochannel 250A and a microchannel 250B separated by amembrane 208, without operating chambers on the sides. In thisembodiment, since the membrane 208 does not need to be mechanicallymodulated, the membrane can be rigid or at least partially flexible. Inone embodiment, the membrane 208 is rigid.

By way of example only, some embodiments of the devices describedherein, e.g., as shown in FIG. 2G, can be used to model at least aportion of a human airway (e.g., a human small airway or large airway).In order to mimic the tissue structure of an airway,anatomically-relevant dimensions should be used. As human lung smallairway is anatomically defined as an airway with a radius of less thanor equal to 1 mm, in one embodiment, the device to mimic at least aportion of a human small airway (small airway-on-a-chip) is designedsuch that the mesochannel (the “airway lumen” channel) has a heightcorresponding to the radius of small airway in vivo (e.g., less than orequal to about 1 mm). In one embodiment, the small airway-on-a-chip hasa mesochannel with a height of about 1 mm.

It should be noted that although the central and operating channels 230,252 are shown to have substantially square or rectangular crosssections, other cross-sectional shapes such as circular, oval, andhexagonal, can also be used. In some embodiments, the central channel230 can have a polygonal cross-section (e.g., U-shaped,polygonal-shaped, or comb-shaped). By way of example only, as shown inFIGS. 22A-22B, the central channel 2230 can have a substantiallyU-shaped cross-section. The U-shaped cross-section can be formed, forexample by having a partition wall 2252 or 2262 disposed in a widermesochannel 2250 (2250A, 2250B) and microchannel 2260 (2260A, 2260B),respectively, wherein the partition wall is disposed traverse to theplane 2208P. Thus, the wider mesochannel 2250 can be divided into two ormore smaller mesochannels (2250A, 2250B); and/or the wider microchannel2260 can be divided into two or more smaller microchannels (2260A,2260B). FIG. 22A illustrates a device 2200A comprising at least onemesochannel 2250 separated from at least two microchannels 2260A and2260B by a membrane 2208. FIG. 22B illustrates a device 2200B comprisingat least two mesochannels 2250A and 2250B separated from at least onemicrochannel 2260 by a membrane 2208. Accordingly, in some embodiments,at least one or more (e.g., 1, 2, 3, 4, or more) partition walls can bedisposed in the wider mesochannel and/or the microchannel to formmultiple sub-channels therein (i.e., to form smaller mesochannels and/ormicrochannels), wherein the partition walls are disposed transverse tothe plane 2208P. The partitions walls can have substantially the sameheight as the mesochannel or microchannel, depending on where they aredisposed. The partition wall can have any thickness as long as they arestructurally stable. The partition walls can form a fluidic seal withthe membrane 2208 such that there is no fluid communication between thesub-channels (e.g., 2250A and 2250B; 2260A and 2260B). In theseembodiments, different cell types cultured in separate sub-channels onone surface of the membrane can interact with the same cell type(s) onthe other surface of the membrane. The same or different fluids can beintroduced into individual sub-channels.

In these embodiments where at least one partition wall is present, thewidth of the sub-channels (e.g., 2250A and 2250B; or 2260A and 2260B) issmaller than the width of the central channel as described earlier. Insome embodiments, the width of the sub-channels can be in a rangebetween 50 microns and 10 mm, or between 100 microns and 5 mm, orbetween 200 microns and 1500 microns, or between 400 microns and 1000microns, or between 50 and 2000 microns. Each sub-channel can have thesame or different width.

In some embodiments, the device 200 can have more or less than twooperating channels 252 and/or more or less than two central channels250A, 250B in accordance with an embodiment.

In accordance with some embodiments of the invention, the device can beplaced in or secured to a cartridge. In accordance with some embodimentsof the invention, the device can be integrated into a cartridge and forma monolithic part. Some examples of a cartridge are described in U.S.Application No. 61/856,876 filed Jul. 22, 2013; U.S. ProvisionalApplication No. 61/696,997, filed on Sep. 5, 2012 and No. 61/735,215,filed on Dec. 10, 2012, contents of each application is incorporatedherein by reference in its entirety. The cartridge can be placed intoand removed from a cartridge holder that can establish fluidicconnections upon or after placement and optionally seal the fluidicconnections upon removal. In accordance with some embodiments of theinvention, the cartridge can be incorporated or integrated with at leastone sensor, which can be placed in direct or indirect contact with afluid flowing through a specific portion of the cartridge duringoperation. In accordance with some embodiments of the invention, thecartridge can be incorporated or integrated with at least one electricor electronic circuit, for example, in the form of a printed circuitboard or flexible circuit. In accordance with some embodiments of theinvention, the cartridge can comprise a gasketing embossment to providefluidic routing.

In accordance with some embodiments of the invention, the cartridgeand/or the device described herein can comprise a barcode. The barcodecan be unique to types and/or status of the cells present on themembrane. Thus, the barcode can be used as an identifier of each deviceadapted to mimic function of at least a portion of a specific tissueand/or a specific tissue-specific condition. Prior to operation, thebarcode of the cartridge can be read by an instrument so that thecartridge can be placed and/or aligned in a cartridge holder for properfluidic connections and/or proper association of the data obtainedduring operation of each device. In accordance with some embodiments ofthe invention, data obtained from each device include, but are notlimited to, cell response, immune cell recruitment, intracellularprotein expression, gene expression, cytokine/chemokine expression, cellmorphology, functional data such as effectiveness of an endothelium as abarrier, concentration change of an agent that is introduced into thedevice, or any combinations thereof.

In accordance with some embodiments of the invention, the device can beconnected to the cartridge by an interconnect adapter that connects someor all of the inlet and outlet ports of the device to microfluidicchannels or ports on the cartridge. Some examples interconnect adaptersare disclosed in U.S. Provisional Application No. 61/839,702, filed onJun. 26, 2013, and the International Patent Application No.PCT/US2014/044417 filed Jun. 26, 2014, the contents of each of which arehereby incorporated by reference in their entirety. The interconnectadapter can include one or more nozzles having fluidic channels that canbe received by ports of the device described herein. The interconnectadapter can also include nozzles having fluidic channels that can bereceived by ports of the cartridge.

In accordance with some embodiments described herein, the interconnectadaptor can comprise a septum interconnector that can permit the portsof the device to establish transient fluidic connection duringoperation, and provide a sealing of the fluidic connections when not inuse, thus minimizing contamination of the cells and the device. Someexamples of a septum interconnector are described in U.S. ProvisionalApplication No. 61/810,944 filed Apr. 11, 2013, the content of which isincorporated herein by reference in its entirety.

Membrane:

The membrane can be porous (e.g., permeable or selectively permeable),non-porous (e.g., non-permeable), rigid, flexible, elastic or anycombinations thereof. Accordingly, the membrane 208 can have a porosityof about 0% to about 99%. As used herein, the term “porosity” is ameasure of total void space (e.g., through-holes, openings, interstitialspaces, and/or hollow conduits) in a material, and is a fraction ofvolume of total voids over the total volume, as a percentage between 0and 100% (or between 0 and 1). A membrane with substantially zeroporosity is non-porous or non-permeable.

As used interchangeably herein, the terms “non-porous” and“non-permeable” refer to a material that does not allow any molecule orsubstance to pass through.

In some embodiments, the membrane can be porous and thus allowmolecules, cells, particulates, chemicals and/or media to migrate ortransfer between the mesochannel 250A and the microchannel 250B via themembrane 208 from the mesochannel 250A to the microchannel 250B or viceversa.

As used herein, the term “porous” generally refers to a material that ispermeable or selectively permeable. The term “permeable” as used hereinmeans a material that permits passage of a fluid (e.g., liquid or gas),a molecule, a whole living cell and/or at least a portion of a wholeliving cell, e.g., for formation of cell-cell contacts. The term“selectively permeable” as used herein refers to a material that permitspassage of one or more target group or species, but act as a barrier tonon-target groups or species. For example, a selectively-permeablemembrane can allow passage of a fluid (e.g., liquid and/or gas),nutrients, wastes, cytokines, and/or chemokines from one side of themembrane to another side of the membrane, but does not allow wholeliving cells to pass therethrough. In some embodiments, aselectively-permeable membrane can allow certain cell types to passtherethrough but not other cell types.

The permeability of the membrane to individual matter/species can bedetermined based on a number of factors, including, e.g., materialproperty of the membrane (e.g., pore size, and/or porosity), interactionand/or affinity between the membrane material and individualspecies/matter, individual species size, concentration gradient ofindividual species between both sides of the membrane, elasticity ofindividual species, and/or any combinations thereof.

A porous membrane can have through-holes or pore apertures extendingvertically and/or laterally between two surfaces of the membrane (FIG.2B), and/or a connected network of pores or void spaces (which can, forexample, be openings, interstitial spaces or hollow conduits) throughoutits volume. The porous nature of the membrane can be contributed by aninherent physical property of the selected membrane material, and/orintroduction of conduits, apertures and/or holes into the membranematerial.

In some embodiments, a membrane can be a porous scaffold or a mesh. Insome embodiments, the porous scaffold or mesh can be made from at leastone extracellular matrix polymer (e.g., but not limited to collagen,alginate, gelatin, fibrin, laminin, hydroxyapatite, hyaluronic acid,fibroin, and/or chitosan), and/or a biopolymer or biocompatible material(e.g., but not limited to, polydimethylsiloxane (PDMS), polyurethane,styrene-ethylene-butylene-styrene (SEBS), poly(hydroxyethylmethacrylate)(pHEMA), polyethylene glycol, polyvinyl alcohol and/or any biocompatiblematerial described herein for fabrication of the device body) by anymethods known in the art, including, e.g., but not limited to,electrospinning, cryogelation, evaporative casting, and/or 3D printing.See, e.g., Sun et al. (2012) “Direct-Write Assembly of 3DSilk/Hydroxyapatite Scaffolds for Bone Co-Cultures.” Advanced HealthcareMaterials, no. 1: 729-735; Shepherd et al. (2011) “3D MicroperiodicHydrogel Scaffolds for Robust Neuronal Cultures.” Advanced FunctionalMaterials 21: 47-54; and Barry III et al. (2009) “Direct-Write Assemblyof 3D Hydrogel Scaffolds for Guided Cell Growth.” Advanced Materials 21:1-4, for examples of a 3D biopolymer scaffold or mesh that can be usedas a membrane in the device described herein.

In some embodiments, a membrane can comprise an elastomeric portionfabricated from a styrenic block copolymer-comprising composition, e.g.,as described in the U.S. Provisional Application No. 61/919,181 filedDec. 20, 2013 and the corresponding PCT International Application No.PCT/US2014/071611, entitled “Organomimetic devices and methods of useand manufacturing thereof” filed concurrently with the currentapplication on Dec. 19, 2014, can be adopted in the devices describedherein, the contents of each of which are incorporated herein byreference. In some embodiments, the styrenic block copolymer-comprisingcomposition can comprise SEBS and polypropylene.

In some embodiments, a membrane can be a hydrogel or a gel comprising anextracellular matrix polymer, and/or a biopolymer or biocompatiblematerial. In some embodiments, the hydrogel or gel can be embedded witha conduit network, e.g., to promote fluid and/or molecule transport.See, e.g., Wu et al. (2011) “Omnidirectional Printing of 3DMicrovascular Networks.” Advanced Materials 23: H178-H183; and Wu et al.(2010) “Direct-write assembly of biomimetic microvascular networks forefficient fluid transport.” Soft Matter 6: 739-742, for example methodsof introducing a conduit network into a gel material.

In some embodiments, a porous membrane can be a solid biocompatiblematerial or polymer that is inherently permeable to at least onematter/species (e.g., gas molecules) and/or permits formation ofcell-cell contacts. In some embodiments, through-holes or apertures canbe introduced into the solid biocompatible material or polymer, e.g., toenhance fluid/molecule transport and/or cell migration. In oneembodiment, through-holes or apertures can be cut or etched through thesolid biocompatible material such that the through-holes or aperturesextend vertically and/or laterally between the two surfaces of themembrane 208A and 208B. It should also be noted that the pores canadditionally or alternatively incorporate slits or other shapedapertures along at least a portion of the membrane 208 which allowcells, particulates, chemicals and/or fluids to pass through themembrane 208 from one section of the central channel to the other.

The pores of the membrane (including pore apertures extending throughthe membrane 208 from the top 208A to bottom 208B surfaces thereofand/or a connected network of void space within the membrane 208) canhave a cross-section of any size and/or shape. For example, the porescan have a pentagonal, circular, hexagonal, square, elliptical, oval,diamond, and/or triangular shape.

The cross-section of the pores can have any width dimension providedthat they permit desired molecules and/or cells to pass through themembrane. In some embodiments, the pore size can be selected to permitpassage of cells (e.g., immune cells and/or cancer cells) from one sideof the membrane to the other. In some embodiments, the pore size can beselected to permit passage of nutrient molecules. When cells arecultured on the membrane at an air-liquid interface, the pore size ofthe membrane should be big enough to provide the cells sufficient accessto nutrients present in the “liquid” channel (or the microchannel). Insome embodiments, the width dimension of the pores can be selected topermit molecules, particulates and/or fluids to pass through themembrane 208 but prevent cells from passing through the membrane 208. Insome embodiments, the width dimension of the pores can be selected topermit cells, molecules, particulates and/or fluids to pass through themembrane 208. Thus, the width dimension of the pores can be selected, inpart, based on the sizes of the cells, molecules, and/or particulates ofinterest. In some embodiments, the width dimension of the pores (e.g.,diameter of circular pores) can be in the range of 0.01 microns and 20microns, or in one embodiment, approximately 0.1-10 microns, orapproximately 7-10 microns. However, in some embodiments, the widthdimension can be outside of the range provided above. In someembodiments, the membrane 208 has pores or apertures larger thantraditional molecular/chemical filtration devices, which allow cells aswell as molecules to migrate across the membrane 208 from one channelsection (e.g. 250A) to the other channel section (e.g. 250B) or viceversa. In one embodiment, the width dimension of the pores can beselected such that a selected type of cells, but not all different typesof the cells present on the membrane, can migrate through the pores.

In some embodiments where the porous membrane comprise through-holes orpore apertures, the pore apertures can be randomly or uniformlydistributed (e.g., in an array or in a specific pattern, or in agradient of pore sizes) on the membrane. In one embodiment, the poreapertures are hexagonally arranged on the membrane. In one embodiment,at least some or all of the pore apertures are equidistant to eachneighboring pore aperture. In this embodiment, at least some or all ofthe pore apertures can have a center-to-center pore spacing of about 1μm to about 1000 μm, or about 10 μm to about 500 μm, or about 20 μm toabout 100 μm. In one embodiment, at least some or all of the poreaperture can have a center-to-center pore spacing of about 20 μm toabout 50 μm. The spacing between pores can vary, e.g., with cell sizes.Without wishing to be bound by theory, larger pore spacing can be usedfor bigger cells, e.g., epithelial cells, and similarly, smaller porespacing can be used for smaller cells.

In an embodiment, the porous membrane 208 can be designed or surfacepatterned to include micro and/or nanoscopic patterns therein such asgrooves and ridges, whereby any parameter or characteristic of thepatterns can be designed to desired sizes, shapes, thicknesses, fillingmaterials, and the like.

The surface area of the membrane exposed to the mesochannel 250A and themicrochannel 250B can vary, e.g., depending on the physiologicalratio(s) of the surface area to the volume of an organ or a tissue to bemodeled, volume of the mesochannel and/or microchannel, cell analysisand/or detection methods, and any combinations thereof. A properratio(s) of the surface area of the membrane exposed to the mesochanneland/or microchannel to the volume of the mesochannel and/or microchannelcan ensure that the device can function more like an in vivo organ ortissue, which can in turn allow for in vitro results to be extrapolatedto an in vivo system. In some embodiments, the surface area of themembrane exposed to the mesochannel 250A and the microchannel 250B canbe configured to satisfy the physiological ratio(s) of the surface areato the volume of an organ or tissue to be modeled. In some embodiments,the surface area of the membrane can be configured to provide asufficient space for cell culture, e.g., such that a sufficient amountof cellular materials (e.g., protein, RNA, secreted cytokines and/orchemokines) can be collected for analysis, e.g., using quantitative PCR,ELISA, sequencing and/or mass spectroscopy. In some embodiments, thesurface area of the membrane can be configured to provide a sufficientspace for examination and/or monitoring of cell behavior, e.g., but notlimited to, immune cell recruitment and/or extravasation.

The membrane 208 can have any thickness provided that the selectedthickness does not significantly affect cell behavior and/or response.For example, in some embodiments, the thickness of the membrane can beselected such that it does not significantly slow down or inhibittransmigration of cells (e.g., immune cells and/or cancer cells) fromone side of the membrane to the other; and/or it does not affect accessof cells growing in the “airway lumen” channel” to nutrients in the“blood vessel” channel. In some embodiments, the thickness of themembrane 208 can range between 70 nanometers and 100 microns, or between1 microns and 100 microns, or between 10 and 100 microns. In oneembodiment, the thickness of the membrane 208 can range between 10microns and 50 microns. While the membrane 208 generally have a uniformthickness across the entire length or width, in some embodiments, themembrane 208 can be designed to include regions which have lesser orgreater thicknesses than other regions in the membrane 208. Thedecreased thickness area(s) 209 can run along the entire length or widthof the membrane 208 or can alternatively be located at only certainlocations of the membrane 208. The decreased thickness area can bepresent along the bottom surface of the membrane 208 (i.e. facingmicrochannel 250B), or additionally/alternatively be on the opposingsurface of the membrane 208 (i.e. facing microchannel 250A). It shouldalso be noted that at least portions of the membrane 208 can have one ormore larger thickness areas relative to the rest of the membrane, andcapable of having the same alternatives as the decreased thickness areasdescribed above.

The membrane 208 can be rigid or flexible. In some embodiments, themembrane can be made of a rigid material, e.g., but not limited topolycarbonate. In some embodiments, the membrane can be made of flexiblematerial, e.g., a polydimethylsiloxane (PDMS) or any other polymericcompound or material. For instance, the membrane 208 can be made ofpolyimide, polyester, polycarbonate, cyclicolefin copolymer,polymethylmethacrylate, nylon, polyisoprene, polybutadiene,polychlorophene, polyisobutylene, poly(styrene-butadiene-styrene),nitriles, the polyurethanes and the polysilicones. GE RTV 615, avinyl-silane crosslinked (type) silicone elastomer (family) can be used.Polydimethysiloxane (PDMS) membranes are available HT-6135 and HT-6240membranes from Bisco Silicons (Elk Grove, Ill.) and are useful inselected applications. The choice of materials typically depends uponthe particular material properties (e.g., solvent resistance, stiffness,fluid permeability, and/or temperature stability) required for theapplication being conducted. Additional elastomeric materials that canbe used in the manufacture of the components of the microfluidic devicesdescribed in Unger et al. (2000 Science 288:113-116). Some elastomers ofthe present devices are used as diaphragms and in addition to theirstretch and relax properties, are also selected for their porosity,permeability, chemical resistance, and their wetting and passivatingcharacteristics. Other elastomers are selected for their thermalconductivity. Micronics Parker Chomerics Thermagap material61-02-0404-F574 (0.020″ thick) is a soft elastomer (<5Shore A) needingonly a pressure of 5 to 10 psi to provide a thermal conductivity of 1.6W/m-° K. Deformable films, lacking elasticity, can also be used in themicrofluidic device. One can also use silk, ECM gels with or withoutcrosslinking as other such suitable materials to make the devices andmembranes as described herein.

The device 200 described herein can be used for various applicationsranging from studying different cell processes, e.g., but not limitedto, ciliary clearance of particulates, epithelial differentiation andcytokine production, inflammatory response to developing relevantdisease models, e.g., but not limited to, asthma, COPD, pulmonaryhypertension, radiation induced injury, cystic fibrosis, and airbornediseases such as viral infection or bacterial infection. The device 200or 201 can be used with or without underlying endothelium, differentimmune cell types or white blood cell types, smooth muscle cells,fibroblasts, etc. For example, in one application, the inventors havefor the first time demonstrated differentiation of human primary airwayepithelial cells in a microfluidic platform using one embodiment of thedevice described herein, e.g., as shown in FIG. 2D or FIG. 2G.

In one embodiment, the membrane 208 can be subjected to physiologicalmechanical strain generated by cyclic stretching of the membrane 208and/or the flow of biological fluids (e.g. air, mucus, blood, culturemedium) in either one or both of the mesochannel and microchannel torecapitulate the native microenvironment of the airway and optionalunderlying capillaries. In an embodiment, the culture conditions ofcells upon the membrane 208 can be optimized under extracellular matrix(ECM) coating, media perfusion, or cyclic mechanical strain to maintainmorphological and functional characteristics of the co-cultured cellsand to permit their direct cellular interaction across the membrane 208.The device 200 would thus permit long-term cell culture and optionaldynamic mechanical stretching of adjacent monolayers of airwayepithelial or endothelial cells grown on the membrane at the same time.

The cells on the membrane 208 can display at least one characteristiccorresponding to a pre-determined physiological endpoint. As usedherein, the term “physiological endpoint” refers to a pre-determinedstate of cells desired to be reach at a certain time point. The cellscan be maintained at the same physiological endpoint in the devices overtime, or they can reach a different physiological endpoint in thedevices at a later time point. Examples of the pre-determinedphysiological endpoint can include, but are not limited to, a maturestate, a differentiated state, a precursor state, a stratified state, apseudo-stratified state, a confluency state, an inflamed state, aninfected state, a stimulated state, an activated state, an inhibitorystate, a normal healthy state, a disease-specific state, a pre-diseasestate, a distressed state, a growth state, a migratory state, athree-dimensional state, or any combinations thereof.

As used herein, the term “precursor state” refers to a cell having acapability to differentiate into a mature cell. Thus, a precursor staterefers to a cell which is partially or fully undifferentiated. In someembodiments, a cell at a precursor state can include apartially-undifferentiated cell that is capable of de-differentiating toa more primitive state. In some embodiments, the term “precursor state”can refer to a progenitor cell or a stem cell.

As used herein, the term “mature state” refers to a fully differentiatedcell of a specific tissue. A mature cell is neither a fetal cell nor anembryonic cell, and is not of the gamete lineage.

As used herein, the term “differentiated state” refers to a cell that ispartially or fully differentiated to a tissue-specific cell. Afully-differentiated cell can be considered as a cell in a mature stateas defined herein. In some embodiments, the differentiated cells canform a stratified structure. In some embodiments, the differentiatedcells can form a 3-D structure.

As used herein, the term “stratified state” refers to cellssubstantially arranged in more than one layer, e.g., 2 layers, 3 layers,4 layers, or more.

As used herein, the term “pseudo-stratified state” refers to cellspresent in a single layer, but when they are visualized byimmunostaining they appear as if they form multiple layers. For example,a pseudostratified epithelium is a type of epithelium that, thoughcomprising only a single layer of cells, has its cell nuclei positionedat different levels, thus creating an illusion of cellularstratification.

As used herein, the term “confluency state” refers to a state wherecomplete or almost complete (at least approximately 50-60% coverage)coverage of a surface area by the cells (e.g., available membranesurface area allowed for cell proliferation).

As used herein, the term “inflamed state” refers to cells showing atleast one phenotype associated with inflammation. Exemplary phenotypesassociated with inflammation include, but are not limited to, attachmentand recruitment of immune cells (e.g., but not limited to neutrophils,monocytes, lymphocytes, dendritic cells and immature macrophages),presence or increased expression of inflammation-associated secretedcytokines/chemokines and/or intracellular molecules, decreased number ofciliated cells, abnormal cilia morphology, increased proportion ofgoblet cells, increased mucus secretion, abnormal cilia beatingfrequency, and any combinations thereof.

As used herein, the term “infected state” refers to cells showing atleast one phenotype associated with microbial infection, e.g., but notlimited to, viral infection, bacterial infection, fungus infection,parasitic infection, and/or any combinations thereof. Exemplaryphenotypes associated with microbial infection, include, but are notlimited to, presence of microbial proteins (e.g., viral/bacterialproteins) in an infected cell, damage to an infected cell's epithelium,elevated levels of cytokines/chemokines such as CXCL10 or IL8 secretedby an infected cell, presence of a cellular antimicrobial protein (e.g.,antiviral protein such as MX proteins), microbial replication ineffluents from the mesochannel/microchannel, and any combinationsthereof.

As used herein, the term “activated state” refers to cells having atleast one cellular process (e.g., but not limited to, migrationpotential, cell proliferation, protein synthesis and/or cytokinesecretion) in an active state. The cellular process can be effected, forexample, by a change in at least one gene expression and/orphosphorylation/dephosphorylation of at least one protein.

As used herein, the term “inhibitory state” refers to cells having atleast one cellular process (e.g., but not limited to, migrationpotential, cell proliferation, protein synthesis and/or cytokinesecretion) in an inhibitory state. The cellular process can be effected,for example, by a change in at least one gene expression and/orphosphorylation/dephosphorylation of at least one protein.

As used herein, the term “stimulated state” refers to a state of cellsthat are responsive to a condition-inducing agent exposed to them. Asused herein, the term “condition-inducing agent” refers to any agentthat can cause a cell to display a phenotype that is deviated from abasal state (without exposure to the condition-inducing agent). Thecondition-inducing agent can provoke a beneficial or adverse effect suchas cytotoxic effect on the cells. In some embodiments Examples of acondition-inducing agent can include, but are not limited to,environmental agents such as radiation (e.g., gamma radiation) andmechanical stress (e.g., fluid shear stress); proteins, peptides,nucleic acids, antigens, cytokines, growth factors, toxins, cells(including prokaryotic and eukaryotic cells such as virus, bacteria,fungus, parasites, and mammalian cells), particulates (e.g., smokeparticles or asbestos), particles (e.g., nanoparticles ormicroparticles, magnetic particles), small molecules, biologics, and anycombinations thereof. Thus, a stimulated state can encompass a maturestate, a differentiated state, a precursor state, a stratified state, apseudo-stratified state, an inflamed state, an infected state, anactivated state, a disease-specific state, and any combinations thereof.

As used herein, the term “normal healthy state” refers to a statewithout any symptoms of any diseases or disorders, or not identifiedwith any diseases or disorders, or not on any physical, chemical and/orbiological treatment, or a state that is identified as healthy byskilled practitioners based on microscopic examinations.

As used herein, the term “disease-specific state” refers to a state ofcells that recapitulates at least one characteristic associated with adisease, disorder or an injury, or different stages thereof. In someembodiments, the term “disease-specific state” can refer to a specificstage or grade of a disease, disorder or an injury. Examples ofdiseases, disorders, or injuries can be related to any organ or tissue,e.g., but not limited to, lung, brain, nerve network,blood-brain-barrier, vascular, kidney, liver, heart, spleen, pancreas,ovary, testis, prostate, skin, eye, ear, skeletal muscle, colon,intestine, and esophagus. In some embodiments, the disease-specificstate can be associated with a lung disease, e.g., but not limited to,asthma, chronic obstructive pulmonary disease (COPD), pulmonaryhypertension, radiation induced injury, cystic fibrosis, or anycombinations thereof. In some embodiments, the disease-specific statecan be associated with an intestinal disease, e.g., but not limited to,inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiacdisease, angiodysplasia, appendicitis, bowel twist, chronic functionalabdominal pain, coeliac disease, colorectal cancer, diverticulardisease, endometriosis, enteroviruses, gastroenteritis, Hirschsprung'sdisease, ileitis, irritable bowel syndrome, polyp, pseudomembranouscolitis, or any combinations thereof. In some embodiments, thedisease-specific state can include a specific stage of a cancer.

The cell in a disease-specific state can be obtained either from abiopsy of a patient carrying the disease, disorder or an injury, or byinducing a normal healthy cell with a condition-inducing agent (e.g., anenvironmental agent such as radiation; a chemical or biological agent,e.g., but not limited to, cytokines described herein and/or pathogens)that is known to induce the cell to acquire at least one characteristicassociated with the disease, disorder, or injury.

As used herein, the term “growth state” refers to a state at which cellsare growing in size and/or in numbers. In some embodiments, the cells ata growth state are undergoing an exponential growth.

As used herein, the term “migratory state” refers to cells having oradopting at least one or more migratory phenotypes, e.g., but notlimited to, disruption of cadherens junctions (e.g., E-cadherinjunctions); increased metalloproteinase expression; loss of anapico-basal polarity, a spindle-shaped morphology, cell-cell interactionthrough focal points, and any combinations thereof. In some embodiments,the migratory state can include an epithelial-mesenchymal transition ortransformation (EMT), which is a process by which epithelial cells losetheir cell polarity and cell-cell adhesion, and gain migratoryproperties to become mesenchymal cells. EMT occurs in variousdevelopmental processes including mesoderm formation and neural tubeformation. EMT also occurs in wound healing, in organ fibrosis and inthe initiation of metastasis for cancer progression. In someembodiments, the devices described herein can be used to modelmetastasis, wherein at least some cancer cells undergo EMT and becomemigratory and migrate from one side of the membrane (where the tumorcells reside) to the other, which is the “blood vessel” channel.

As used herein, the term “metamorphosing state” refers to a tissue(e.g., a group of cells) being readily capable of or undergoingmetamorphosis or a developmental transition. In some embodiments, ametamorphosing state refers to an embryonic tissue undergoing induction(e.g., epithelial-mesenchyme interface transforming into a fully orpartially-developed specific tissue, e.g., tooth, bone or epithelialgland). In some embodiments, a metamorphosing state refers to an insecttissue undergoing metamorphosis or any whole tissue undergoing a wholedevelopmental transition.

As used herein, the term “three-dimensional state” refers to arrangementof cells in a three-dimensional structure. By way of example only,intestinal epithelial cells grow into folds and form villi in form oftubular projections.

The device described herein can be utilized to grow and culture cells toreach a pre-determined physiological endpoint by optimizing cell cultureconditions. Cell culture conditions that can be optimized include, butare not limited to, seeding density, cell source and/or type, supportingcells, composition of the media, flow rate of air and/or media, presenceor absence of an air-liquid interface, requirement of mechanicalstimulation (e.g., induced by the membrane movement), membrane surfaceproperties, dimensions of the mesochannel and/or microchannel, or anycombinations thereof. The pre-determined physiological endpoint can bedetected by cell morphology and/or the presence of at least one markerassociated with the pre-determined physiological endpoint, which isfurther illustrated in the example below.

Optimization of Cell Culture Conditions to Reach a Pre-DeterminedPhysiological Endpoint:

As discussed above, a number of cell culture condition parameters can beoptimized in a device described herein for different pre-determinedphysiological endpoints. Exemplary cell culture condition parametersinclude, but are not limited to, cell-related parameters (e.g., cellsources, cell types, supporting cells, seeding density, and degree ofconfluency); culture medium-related parameters (e.g., composition orformulation of culture media); microenvironment-related parameters(e.g., flow rates of air and/or media, presence or absence of anair-liquid interface, mechanical stimulation requirement, membranesurface properties, and dimensions of the mesochannel and/ormicrochannel), and any combinations thereof.

Cell-Related Parameters:

Cells used in the device can be primary cells (e.g., any cells obtaineddirectly from a living tissue, e.g., a biopsy material, of a human or ananimal, which include, but are not limited to normal healthy cells, anddisease-specific cells), immortalized or established cell lines, stemcells (e.g., embryonic stem cells, fetal stem cells, adult stem cells,stem cells derived from bone marrow, cord blood, and/or an amnioticfluid, induced pluripotent stem cells, and patient-specific stem cells),and/or modified cells.

In some embodiments, the cells used in the device described herein cancomprise primary cells. For example, normal healthy cells can beobtained from one or more healthy donors. Disease-specific cells can beobtained from one or more patients diagnosed with the specific disease.For example, asthmatic, chronic obstructive pulmonary disease (COPD) andcystic fibrosis (CF)-associated airway cells can be obtained from one ormore asthmatic, COPD and CF patients, respectively.

In some embodiments, the phenotype and/or behavior of the cells can bemodified with a condition-inducing agent described herein. For example,normal healthy cells can be transformed to behave like disease-specificcells phenotypically and/or morphologically by stimulating the normalhealthy cells with an agent known to induce symptom(s) of a specificdisease in the cells. In one embodiment, cigarette smoke can be used tostimulate normal healthy cells for inducing chronic obstructivepulmonary disease (COPD) phenotype. In another embodiment,asthmatic-like cells can be derived from normal healthy cells byinducing inflammation in the normal healthy cells, e.g., by exposure toa pro-inflammatory factor described herein, e.g., but not limited to,TNF-alpha; by stimulation of normal cells with an allergen (e.g., housedust mite); and/or by stimulation with TH2 cytokines such as IL-13.

In some embodiments, the cells used in the device described herein canbe genetically modified, e.g., by silencing one or more genes, orover-expressing one or more genes. Exemplary methods of gene silencinginclude, but are not limited to, RNA interference (e.g., but not limitedto small interfering RNA (siRNA), microRNA (miRNA), and/or short hairpinRNA (shRNA)), antisense oligonucleotides, ribozymes, triplex formingoligonucleotides, and the like. Alternatively or additionally, the cellscan be labeled with a detectable reporter (e.g., an optical reportersuch as a fluorescent molecule, and/or a protein tag). By way of exampleonly, CF-associated airway cells can be derived from normal healthycells by a knock-out or silencing of the cystic fibrosis transmembraneconductance regulator (CFTR) gene, in which the presence of at least oneor more mutations is known to cause CF. Methods for gene silencing isknown in the art. For example, a CFTR-targeting shRNA, siRNA, antisenseoligonucleotide, ribozyme, and/or triplex forming oligonucleotide can beintroduced into normal healthy airway cells (e.g., primary cells), e.g.,by a lentivirus system, in order to silence the CFTR gene, which can inturn result in a CF phenotype in the normal healthy cells.

Different cell types can be appropriately selected in accordance with atissue and/or its function to be mimicked. By way of example only, lungalveolar cells can be selected for use in a device described herein tosimulate a microenvironment in a portion of a lung air sac duringbreathing; while airway or bronchial epithelial cells can be used tosimulate a microenvironment in an airway (e.g., a small airway) orbronchus during breathing. Other tissue-specific cells such as heartcells (e.g., but not limited to, cardiac muscle cells, connective tissuecells, aorta cells, atrial cells, ventricular cells, and heart valveinterstitial cells), gut cells (e.g., but not limited to, esophaguscells, stomach cells, intestine cells, and colon cells), liver cells(e.g., but not limited to, karat parenchymal cells, and non-parenchymalcells such as sinusoidal hepatic endothelial cells, Kupffer cells andhepatic stellate cells), and skin cells (e.g., but not limited to,keratinocytes, fibroblasts, adipocytes, connective tissue cells, dermalcells, epidermal cells, and/or gland cells) can be used in the devicesdescribed herein to simulate a portion of a corresponding tissue.Additional cell types of various tissues that can be used in the devicesdescribed herein are described in the section “Cells” below. In someembodiments, stem cells can be used to differentiate into different celltypes.

In some embodiments, supporting cells can be cultured together withsubject cells of interest. As used herein, the term “supporting cells”refers to cells that provide protection, support, chemical signals(e.g., factors secreted by the supporting cells) and/or physical signals(e.g., direct physical contact between the subject cells and thesupporting cells) that can be essential for proper phenotypes and/orfunctions of the subject cells of interest. For example, interstitialcells (e.g., but not limited to fibroblasts and/or smooth muscle cells)can be used as supporting cells for epithelial cells and act as a“feeder” layer for the epithelium. In one embodiment, lung interstitialcells (e.g., fibroblasts and/or smooth muscle cells) can be used assupporting cells for airway epithelial cells.

Seeding density and/or degree of cell confluency can influence cellmorphology and/or their behavior (e.g., but not limited to,proliferation, viability, migration, protein synthesis, and/ordifferentiation). The cell seeding density and/or degree of cellconfluency can be optimized for individual cell types (e.g., cell size,and/or modes of cell signaling such as direct contact, paracrinesignaling, and/or endocrine signaling. For example, cells that requireat least a part of the cell body to be in direct contact withneighboring cells in order to proliferate and remain viable generallyneed to be seeded at a higher cell density, as compared to cells thatcan also rely on paracrine signaling. Accordingly, the seeding densityof cells can range from about 0.01 cell/μm² to about 1 cell/μm², or fromabout 0.05 cell/μm² to about 0.5 cell/μm². Similarly, some cells can begrown a in a sparsely-populated environment, while other cell types canrequire a denser population. Thus, degree of cell confluency can rangefrom about 30% to 100% or about 50% to 100%. In one embodiment, airwaycells can be seeded in the device described herein with a seedingdensity of about 0.1 cell/μm², which can provide about 90-100%confluence.

Culture Medium-Related Parameters:

The formulation of cell culture media can vary with individual celltypes and/or their stages within a cell cycle as different cell typescan require a unique combination and concentrations of nutrients and/orsupplements (e.g., growth factors and/or small molecules such as aminoacids and minerals) during different stages of a cell cycle (e.g.,proliferation vs. differentiation). For example, as shown in Example 1,higher concentration of retinoic acid is used in the culture medium toinduce differentiation of airway cells to ciliated and mucus-secretingcells after the cells have proliferated to reach confluency.Accordingly, one or more cell culture media (or a mix of at least twocell culture media) can be used in the devices described herein toachieve any of the physiological endpoints described herein. In someembodiments, a mix of at least two cell culture media can be used in thedevices described herein to accommodate at least two or more cell typesin a co-culture condition. By way of example only, in a co-culturecondition, epithelial cells (optionally with supporting cells such asfibroblasts and/or smooth muscle cells) can be cultured in themesochannel, while endothelial cells (optionally with supporting cells)can be cultured in the microchannel. Alternatively or additionally,immune cells can be introduced into the microchannel, either with astatic fluid or a flowing fluid.

Cell culture media can comprise amino acids (e.g., but not limited to,alanine, arginine, asparagine, aspartic acid, cystine, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, L-methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine); vitamins (e.g., but not limited to,folic acid, i-inositol, ascorbic acid, biotin, choline chloride,Ca⁺⁺-pantothenate, menadione, niacinamide, nicotinic acid,paraaminobenzoic acid (PABA), pyridoxal, pyridoxine, riboflavin,thiamine-HCl, vitamin A acetate, vitamin B12 and vitamin D2); inorganicsalts (e.g., sodium salts, magnesium salts and calcium salts);transition metals, lipids, peptides, carbohydrates (e.g., glucose),sodium pyruvate, a buffered solution (e.g.,N-{2-hydroxyethyl}piperazine-N′-[2-ethanesulfonic acid] (HEPES) or oneor more other zwitterion buffers), a pH indicator (e.g., phenol red),antibiotics (e.g., penicillin and/or streptomycin), cytokines, hormones(e.g., epinephrine, hydrocortisone and/or insulin), serum, serumalbumin, transferrin, retinoic acid (vitamin A), adenine sulfate, ATP,trace elements (e.g., but not limited to, ions of barium, bromine,cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium,titanium, germanium, molybdenum, silicon, iron, fluorine, silver,rubidium, tin, zirconium, cadmium, zinc and aluminum), deoxyribose,ethanolamine, glutathione, hypoxanthine, linoleic acid, lipoic acid,phosphoethanolamine, putrescine, thymidine, uracil, xanthine, anyart-recognized culture supplements, and any combinations thereof. Eachof these ingredients can be obtained commercially, for example fromSigma (Saint Louis, Mo.).

Cytokines which can be used in the cell culture media include growthfactors such as epidermal growth factor (EGF), acidic fibroblast growthfactor (aFGF), basic fibroblast growth factor (bFGF), hepatocyte growthfactor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growthfactor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor(NGF), platelet-derived growth factor (PDGF), transforming growth factorbeta (TGF-β), vascular endothelial cell growth factor (VEGF),transferrin, various interleukins (such as IL-1 through IL-18), variouscolony-stimulating factors (such as granulocyte/macrophagecolony-stimulating factor (GM-CSF)), various interferons (such as IFN-γ)and other cytokines having effects upon hematopoietic stem cells such asstem cell factor (SCF) and erythropoietin (Epo). These cytokines can beobtained commercially, for example from Life Technologies, Inc.(Rockville, Md.) or R&D Systems (Minneapolis, Minn.), of from Peprotech(Rocky Hill, N.J.) and can be either natural or recombinant. In someembodiments, for culture of a wide variety of mammalian cells, the basalmedia can contain EGF at a concentration of about 0.1-100nanograms/milliliter. In some embodiments, the basal media can containEGF at a concentration of about 1-10 nanograms/milliliter. In someembodiments, the basal media can contain EGF at a concentration of about5-10 nanograms per milliliter. Other cytokines, if used, can be added atconcentrations that are determined empirically or as guided by theestablished cytokine art. See the section “Additional examples ofcytokines” for other cytokines that can be added in the cell culturemedia.

Concentrations of each component of the culture media can be optimizedfor different cell types and physiological endpoints to be achieved. Ingeneral, the components of the culture media can each be independentlypresent in an amount in a range of about 1×10⁻¹⁰ mg/L to about 1×10⁴mg/L. For example, the concentration of amino acids can be in a range ofabout 0.05 mg/L to about 750 mg/L; vitamins in a range of about 0.0005mg/L to about 500 mg/L; inorganic salts in a range of about 1 mg/L toabout 10000 mg/L; trace elements in a range of about 1×10⁻¹⁰ mg/L toabout 0.5 mg/L.

In some embodiments, the cell culture media for use in the devicedescribed herein can comprise one or more ingredients of cell culturemedia described in the International Application Publication Nos.: WO2003/048313; WO 2006/004728; WO 2005/065341; WO 2002/077202; WO2010/096588; WO 2005/095582; and WO 1998015614, the contents of whichare incorporated herein by reference.

In some embodiments, the cell culture medium can comprise blood (e.g.,whole blood, plasma, serum, or any combinations thereof). In oneembodiment, the cell culture medium can comprise blood or bloodcomponents derived from a patient for culturing patient-specific cells.

The media can comprise one or more differentiation agents. As usedherein, the term “differentiation agent” refers to molecule(s) and/orcomposition(s) that can induce differentiation of a stem cell or anundifferentiated or partially differentiated cell to a desired state.This can be useful when stem cells or undifferentiated or partiallydifferentiated cells are used.

Microenvironment-Related Parameters:

In addition to the cell-related and culture medium-related parameters,one or more microenvironment-related parameters (e.g., flow rates of airand/or cell culture media, presence or absence of an air-liquidinterface, mechanical cue, membrane surface properties, and dimensionsof the mesochannel and/or microchannel) can be regulated to achieve anyof the physiological endpoints described herein.

A device having a mesochannel and/or microchannel ofphysiologically-relevant dimensions can be used to provide a simulatedtissue microenvironment, which can, at least in part, regulate celldevelopment to various physiological endpoints defined herein. Forexample, the higher mesochannel can offer a reduced stress environmentand increased overhead space for growth and/or differentiation of cellsthat require low shear and/or more space to form a stratified structure.In one embodiment, the higher mesochannel can be used to permitsufficient overhead space for growth and differentiation of airwayepithelial cells to ciliated and mucus-secreting cells.

In some embodiments, an air-liquid interface can be established in thedevices described herein to mimic the native tissue microenvironment oftissue-specific cells and/or induce differentiation and/or maturation ofthe tissue-specific cells. As used herein, the term “air-liquidinterface” refers to one of the mesochannel and microchannel having airtherein while the remaining channel has a liquid fluid, e.g., cellculture medium and/or blood. There can be substantially no liquid fluidpresent in the “air” channel. However, cells present on the membranefacing the “air” channel can secrete a liquid-like substance, such asmucus, and/or a small amount of a liquid fluid can permeate through themembrane from the “liquid” channel to the “air” channel. In someembodiments, the term “air-liquid interface” refers to substantially noliquid fluid being introduced into one of the mesochannel andmicrochannel, while a liquid fluid is introduced into the remainingchannel. In one embodiment, an air-liquid interface refers to themesochannel having air therein while the microchannel has a liquidfluid, e.g., cell culture medium and/or blood. State another way,substantially no liquid fluid is introduced into the mesochannel, whilea liquid fluid is introduced into the microchannel. For example, anair-liquid interface can be established in the devices described hereinto induce differentiation or maturation of airway epithelial cells (asdescribed below) or skin cells. In other embodiments, the nativemicroenvironment of some tissue-specific cells (e.g., heart cells, livercells and/or gut cells) may not require an air-liquid interface. Inthese embodiments, a liquid fluid, e.g., cell culture medium, can bepresent in both the mesochannel and the microchannel.

Air and/or culture media can be introduced into the appropriate channelsin the devices (e.g., mesochannel and microchannel) as a static fluid(which can be periodically replaced) or a continuous (dynamic) flow.Flow rates of air and/or culture media in the mesochannel and/ormicrochannel can be adjusted independently to reflect the physiologicalvalues specific to a tissue-specific condition or state (e.g., a restingstate vs. an active state, e.g., during exercise; or a normal healthystate vs. a disease-specific state). For example, air flow can becontrolled at a volumetric rate to provide a fluid shear stress of about0 dynes/cm² to about 2000 dynes/cm², or 0.1 dynes/cm² to about 2000dynes/cm². In some embodiments where the device is used to mimicbreathing through an airway and/or a lung, the air flow through themesochannel can be adjusted to have a rate of about 1 μL per breath toabout 50 mL per breath, or about 5 μL per breath to about 25 mL perbreath, or about 10 μL per breath to about 10 mL per breath, or about 25μL per breath to about 1 mL per breath. As used herein in reference tothe device, the term “breath” refers to air flow induced in themesochannel to mimic inspiration and expiration of air in a lung. Theair flow volume and/or rhythm can vary depending on the state of a lungto be mimicked. For example, when stimulating air flow in a lung duringexercise, e.g., running, the volume of air getting into and out of thelungs can increase per breath and unit time.

Culture medium flow rates can be controlled to simulate the flow rate ofblood corresponding to a tissue-specific condition or state (e.g., aresting state vs. an active state, e.g., during exercise; or a normalhealthy state vs. a disease-specific state). In some embodiments, theculture medium flow rates can be provided in a range of about 0 μL/hr toabout 50 mL/hr.

In some embodiments where the cells are exposed to a mechanical stressor strain in their native tissue microenvironment such as a strainproduced by motion associated with breathing, peristalsis or heartbeating, the cells present on the membrane can be subjected to asimulated mechanical strain for development of a pre-determinedphysiological endpoint. The simulated mechanical strain can be producedby modulating the movement of the membrane, which can be parallel toand/or perpendicular to a force/pressure applied to the membrane,including, but are not limited to, stretching, bending, compressing,vibrating, contracting, waving, or any combinations thereof. By way ofexample only, in a pulmonary alveolus, alveolar cells experiencestretching when the alveolus is filled with air during inhalation butrestore to an original state or relaxed state during exhalation in orderto expel carbon-dioxide-rich air. Another example is that esophaguscells or intestinal cells are subjected to a mechanical stress or strainproduced by peristaltic waves occurring in the esophagus, or intestines,respectively. In a heart, the atria and ventricles work together,alternately contracting and relaxing to pump blood through the heart. Inorder to simulate a physiological strain produced by motion associatedwith breathing, peristalsis, or heart beating, the membrane can be, inone embodiment, modulated to stretch and release along the plane, e.g.,by a pneumatic mechanism based on application of a pressure differentialbetween the central channel 230 and the operating channel(s) 252 asshown in FIG. 2D, thereby providing the cells (e.g., alveolar cells,esophagus cells, intestinal cells, atrial myocardial cells, andventricular myocardial cells) with a simulated mechanical cue as theyreside in the native tissue microenvironment.

In some embodiments, the membrane can be treated or coated with celladhesion molecules and/or extracellular matrix molecules to facilitatedevelopment of a pre-determined physiological endpoint. Examples of celladhesion molecules, and/or extracellular matrix molecules include,without limitations, fibronectin, laminin, various collagen types,glycoproteins, vitronectin, elastins, fibrin, proteoglycans, heparinsulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid,integrin-binding peptides such as Arg-Gly-Asp (RGD) peptides, or anycombinations thereof.

By way of example only, utilizing the device described herein to reach adifferentiated or mature state of human epithelial cells, e.g., humanprimary airway epithelial cells, one method (e.g., as shown in FIG. 5A)comprises seeding human cells or human primary cells (e.g., humanprimary airway or bronchial epithelial cells) on the membrane in theupper mesochannel (or an “airway lumen” channel). The cells are culturedin a submerged condition by flowing a culture medium through both themesochannel and the microchannel. In some embodiments, the cells arecultured in a submerged condition until the cells reach a fullconfluence. Then, an air-liquid interface is optionally established byremoving the culture medium from the mesochannel through its outlet. Asthe air-liquid interface can induce differentiation of certaincell-types, e.g., airway epithelial cells, the cells can differentiateafter about 3-4 weeks or longer of culture in the device at theair-liquid interface. A static air flow is generally sufficient toinduce cell differentiation. While not necessary, in some embodiments, adynamic air flow can be induced in the mesochannel during celldifferentiation to improve the cellular function(s) of thedifferentiated epithelial cells (e.g., differentiated airway epithelialcells). For example, a dynamic air flow can improve cilia beatingfrequency, mucous secretion, monolayer barrier function (e.g.,permeability of epithelial layer) and/or surface protein expression ofdifferentiated airway epithelial cells.

However, it should be noted that depending on cell types, an air-liquidinterface is not always necessary for cell differentiation. In theseembodiments, a liquid flow can be maintained in the mesochannel duringcell differentiation. For example, intestinal epithelial cells do notrequire an air-liquid interface to undergo villus differentiation.

In some embodiments, a liquid fluid, e.g., cell growth media, flowingthrough the microchannel can comprise at least onedifferentiation-inducing agent, including, e.g., at least two, at leastthree, at least four, at least five differentiation-inducing agents.

In some embodiments, the cells can require exposure to a mechanicalstrain in order to reach a differentiated or mature state. For example,the cells in the mesochannel can be exposed to a mechanical cyclicstrain (e.g., about 0.1% to about 50%, or about 1% to about 30%, orabout 10% to about 25% at a frequency of about 0 Hz to about 1 Hz, orabout 0.01 Hz to about 1 Hz) by stretching and/or retracting themembrane. In one embodiment, intestinal epithelial cells in themesochannel can be exposed to a cyclic stain (e.g., about 10% at ˜0.15Hz).

In one embodiment, differentiation of airway epithelial cells in adevice described herein comprises fully confluent of epithelial cells,an air-liquid interface induction and a cell-growth medium that supportscell differentiation.

While methods of differentiating airway epithelial cells in transwellsystems has been previously reported, technologies and techniques fordifferentiating primary human airway epithelial cells (e.g., human smallairway epithelial cells) in microfluidic setting have not existed yet.For example, simply transposing the composition of growth medium fromtranswell culture is not sufficient. For example, small airwayepithelial cells cultured in a microfluidic setting with a normal growthmedium as used in the transwell culture exhibit a squamous phenotype(FIG. 17B), which is not same as in vivo morphology. To this end, theinventors surprisingly discovered that increasing retinoic acidconcentration in the growth medium can reverse the squamous phenotypeand restore a normal phenotype as observed in vivo. In one embodiment,the inventors discovered that a physiological airway unit (e.g., smallairway unit) can be formed on the membrane 208 of the device, e.g.,using the device and the method described herein. After exposing theairway epithelial cells (e.g., small airway epithelial cells) to anair-liquid interface in the device described herein, the cells aredifferentiated into a 3-D structure of terminally differentiatedciliated and mucous-secreting (goblet) cells, e.g., as detected byimmunofluorescence microscopy and/or scanning electron microscopy (FIGS.5E-5G and 7A-7B). Differentiated cells exhibit cilia beating and mucussecretion (FIGS. 5E-5G and 7A-7B), and tight barrier function (FIGS. 5Dand 7C). Typical junctional structures can form between thedifferentiated airway epithelial cells (e.g., small airway epithelialcells) on the membrane 208 and fluids as well as ions be transportedacross the membrane 208 between the mesochannel and microchannel 250A,250B. The formation of tight junctions between the differentiated airwayepithelial cells on the membrane 208 can be evaluated usingimmunohistochemical detection of tight junction proteins such as ZO-1,TJP-1 (see FIGS. 5D and 7C).

Depending on the nature and/or properties of the selected body material(at least a portion of the device body that is in contact with thefluid), the composition of the cell-growth medium needs to be optimizedaccordingly. For example, in the case of using PDMS to fabricate thedevice body, concentrations of certain hydrophobic components or factorsin the cell growth medium needs to be increased because they likely getabsorbed on the PDMS surface. In one embodiment, retinoic acid in thecell growth medium is increased, e.g., up to 200 times as compared toother material used, to allow sufficient availability of the retinoicacid for the epithelial cells.

Validation/Quality Control Tests of the Physiological Endpoints:

Cells with different physiological endpoints defined herein (e.g.,precursor cells or non-differentiated cells vs. differentiated or maturecells; or normal healthy cells vs. disease-specific cells) can beidentified by methods and assays known to one of skill in the art. Forexample, a physiological endpoint can be identified based on, but notlimited to, cell function, molecule release from cells, cell morphology,cell metabolism, expression level or presence/absence of a moleculeknown to be associated with the pre-determined physiological endpoint.Cells can be analyzed “on-device” (e.g., cells remain inside themesochannel and/or microchannel during analysis) or some cells can beremoved and analyzed “off-device” (e.g., cells are removed from thedevice for subsequent analysis that is not performed on the device).

In some embodiments, the membrane 208 can be removed from the devicesfor analysis, e.g., immunohistochemical detection, immunofluorescencemicroscopy and/or scanning electron microscopy. In other embodiments,the membrane 208 can be evaluated and analyzed using on-chip detectionmethods, e.g., immunohistochemical detection and/or microscopy. In someembodiments, the entire device including the membrane can be evaluatedand analyzed, e.g., under a microscope.

For example, as described earlier, in contrast to non-differentiatedepithelial cells, differentiated airway cells typically form ciliatedcells, globet cells (mucus-secreting cells) and a tight epithelialbarrier, the phenotypes of each of which can be detected, e.g., bystaining the cells for cilia-associated markers (e.g., but not limitedto β-tubulin IV), goblet cell-associated markers (e.g., but not limitedto MU5AC) and/or tight junction-associated markers (e.g., TJP-1 andZO-1), followed by microscopy imaging (FIGS. 5D-5E, and FIGS. 7A-7C).Alternatively or additionally, cilia beating frequency can be determinedby scanning electron microscopy. The barrier function of adifferentiated epithelium can also be determined by a functional assay,e.g., adding fluorescently-labeled large molecules (e.g., inulin-FITC)into a fluid flowing through the mesochannel and then detecting thepresence of the fluorescently-labeled large molecules in themicrochannel, wherein the no detectable fluorescent signal from themicrochannel is indicative of a functional barrier formed by thedifferentiated epithelium (FIG. 7D).

To determine an inflamed state, cell response to inflammation can bequantified by a functional assay and/or cytokine and/or chemokineexpression analysis. For example, attachment and recruitment of immunecells (e.g., but not limited to neutrophils, monocytes, lymphocytes,dendritic cells and immature macrophages) from a static or flowing fluidin the microchannel (“blood vessel” channel) to the membrane and/orepithelium on the side of the mesochannel can be quantified bymicroscopy, histology, and/or by tracking movement of detectable markers(e.g., fluorescently-labeled immune cells) using, e.g., fluorescenceactivated cell sorter (FACS). Alternatively or additionally, cytokineand/or chemokine expression analysis (including secreted and/orintracellular molecules) can be performed by collecting effluents and/orcells from the mesochannel and/or microchannel and detectinginflammation-associated cytokines and/or chemokines, e.g., bymicroarray, ELISA, immunofluorescence, microscopy, and/or quantitativereal-time polymerase chain reaction (PCR).

Other methods that can be used to determine inflammation include, butare not limited to, monitoring the frequency of cilia beating, e.g., bymicroscopy; measuring mucus clearing speed, e.g., by particle imagevelocimetry; evaluating cilia morphology, e.g., by scanning electronmicroscopy; and/or detecting the presence of mucus-producing cells(globet cells) by morphology through microscopic examination and/orapical secretions. To measure mucus clearing speed, for example,particle image velocimetry, can be used as follows: a fluid (e.g., cellculture media) comprising small detectable beads (e.g., fluorescentbeads of ˜1-˜3 microns) can be introduced into the mesochannel whereciliated cells are growing. Under a microscope (e.g., a fluorescentmicroscope), the mucociliary transport (e.g., characterized by speedand/or direction) can be monitored and/or quantified (e.g., based on aseries of recorded images or movies).

In order to distinguish normal healthy cells from disease-specific cellssuch as airway-associated diseases or disorders, one of skill in the artcan compare and contrast phenotypes of the diseased cells with thenormal healthy cells, thereby identifying distinct features between thenormal healthy cells and the diseased cells. By way of example only, inan asthma disease model, asthmatic airway cells can display at least one(including at least two or more) of the following phenotypes, ascompared to normal healthy airway cells: (i) higher mucus secretion byat least about 10% or more; (ii) higher proportion of globet cells(globet cells metaplasia) by at least about 10% or more; and (iii)decreased number of ciliated cells by at least about 5% or more. In someembodiments, an increase in nitric oxide can be detected in the devicewith asthmatic airway cells, as compared to normal healthy cells. Anyart recognized methods, e.g., ELISA, microscopy, immunofluorescence,and/or PCR, can be used to determine cell morphology and itsbehavior/response. For example, mucus secretion by the airway cells canbe determined by ELISA, immunofluorescence, and/or PCR.

In a chronic obstructive pulmonary disease (COPD) disease model,COPD-associated airway cells can display at least one (including atleast two or more) of the following phenotypes, as compared to normalhealthy airway cells: (i) higher basal secretion of pro-inflammatorycytokines/chemokines, e.g., IL-8, by at least about 10% or more; (ii)increased responsiveness to viral and/or bacterial challenges, whichinclude, e.g., more rapid synthesis and/or secretion of at least onepro-inflammatory cytokine/chemokine by at least about 10% or more; (iii)higher mucin gene expression (e.g., measured by real time quantitativePCR) and/or mucin secretion (e.g., measured by ELISA) by at least about10% or more; and (iv) lower ciliated cell count and/or cilia beatingfrequency by at least about 5%. In some embodiments, the COPD-associatedairway cells can display higher adhesion of alveolar macrophages by atleast about 10%, as compared to normal healthy cells. For example, onecan flow a fluid comprising alveolar macrophages through the mesochanneland/or microchannel and then measure the number of the alveolarmacrophages adhered to the COPD cells, e.g., by microscopy.

The cause of cystic fibrosis (CF) is at least in part related to thepresence of at least one or more mutations in cystic fibrosistransmembrane conductance regulator (CFTR) protein, which can affectfunctioning of the chloride ion channels in the cell membranes. Mutationcan include, but are not limited to replacements, duplications,deletions or shortenings in the CFTR gene. These mutations can result indysfunction, faster degradation, and/or lower expression level of theCFTR protein. See, e.g., Rowe, S. M. et al., “Cystic Fibrosis” N Engl JMed 2005; 352:1992-2001. Some of the CFTR mutations can include, but arenot limited to, (i) ΔF508, a deletion (Δ) of three nucleotides whichresults in a loss of the amino acid phenylalanine (F) at the 508thposition on the protein, resulting in faster degradation of the protein;(ii) G542X; (iii) G551D; (iv) N1303K; and (v) W1282X. Accordingly, in aCF disease model, the CF-associated airway cells can display at leastone (including at least two or more) of the following phenotypes, ascompared to normal healthy airway cells: (i) at least one or moremutations in the cystic fibrosis transmembrane conductance regulator(CFTR) protein as described above. In one embodiment, the CFTR mutationcan include ΔF508; (ii) increased mucus secretion and/or mucus thickness(where mucus thickness can be measured, in one embodiment, bytransmission electron microscopy); and (iii) lower cilia beatingfrequency or in some cases, cilia stop beating (e.g., due to mucusgetting thicker and heavier). The mutation in the CFTR protein can bedetermined, e.g., by sequencing to identify the single nucleotidepolymorphisms (SNPs); by performing a gene and/or transcript expressionanalysis to determine a decreased expression of the CFTR protein; and/orby detecting a decreased functioning of the chloride ion channels in thecell membranes of the CF-associated airway cells.

Introduction of a Gas Flow into the Mesochannel:

In some embodiments, one end of the mesochannel can be adapted to engageto a gas-flow modulation device, which can be used to control the flowof a gas through the mesochannel. In some embodiments, the gas-flowmodulation device can be adapted to provide a directional flow of gas oran alternating flow of gas that can reverse its direction periodically.For example, as shown in FIGS. 13A-13D, at least one end or both ends(e.g., 1310 and/or 1312) of the mesochannel 250A can be adapted toengage to a gas-flow modulation device 1314. In some embodiments, theoutlet 1312 of the mesochannel 250A is adapted to engage to a gas-flowmodulation device 1314. The gas-flow modulation device 1314 can be in aform of any reversibly inflatable or reversibly expandable chamber,which can expand and contract to receive and expel a gaseous fluid(e.g., but not limited to air), respectively. The gas-flow modulationdevice can also allow introduction of a particular sample such aspolluted air, cigarette smoke or air-borne viruses. By way of exampleonly, the gas-flow modulation device 1314 can be in a form of a balloon(FIG. 13C), a drum (FIG. 13D), or a thin-walled tube. The drum as shownin FIG. 13D comprise a flexible diaphragm 1315, which can move outward(inflates—away from the inflow direction) and inward (deflates—towardthe inflow direction) to accumulate and expel a gaseous fluid (e.g.,air), respectively.

In some embodiments, the inlet 1310 of the mesochannel 250A can beadapted to engage to a gas-flow generator 1316, e.g., but not limitedto, a ventilator.

In some embodiments, the devices described herein can be used to mimicalternating inspiratory and expiratory airflow during respiration andthus mimic breathing pattern and/or rhythm, e.g., during a restingstate, exercise, stress, or illness, e.g., suffering from a respiratorydisease or distress. For example, the gas-flow modulation device 1314can be configured to create an alternating inspiratory and expiratoryair flow with an average tidal volume ranging from about 10 μL to about5000 μL, or from about 50 μL to about 2500 μL, or from about 75 μL toabout 1000 μL, or from about 100 μL to about 500 μL. The term “tidalvolume” as used herein refers to a volume of air displaced betweeninspiration and expiration when no external pressure is not applied(e.g., to mimic breathing during a resting state). The tidal volume canvary depending on the size of the lung to be mimicked, e.g., a newbornvs. an adult; or a human being vs. a large animal such as an elephant.In some embodiments, the gas-flow modulation device 1314 can beconfigured to create an alternating inspiratory and expiratory air flowwhere a volume of air displaced between inspiration and expiration isgreater or smaller than the tidal volume as defined herein, for example,to mimic breathing during exercise or illness.

In some embodiments, the gas-flow modulation device 1314 can beconfigured to create an alternating inspiratory and expiratory air flowwith a respiratory frequency or rate of about 5 breaths/min to about 100breaths/min, or about 10 breaths/min to about 50 breaths/min.

FIGS. 14A-14B are experimental data showing simulation of respirationusing a gas-flow modulation device in a device according to oneembodiment. One end of the mesochannel (the “airway lumen” channel) ofthe device was adaptably connected to, e.g., a small animal ventilator1316 and attached equipment that can adjust pressure and volume of air,in order to generate air flow. Air was flown from the one end of the“airway lumen” channel, namely “mouth end 1310” into the device—that is“inspiratory flow.” The other end of the “airway lumen” channel, knownas “alveolar end 1312” was adaptably connected to a rubber balloonstructure with compliance and elasticity to help forcing the air out ofthe device—that is “expiratory airflow.” The airflow/breathing can beadjusted in a way to mimic breathing of a human subject in the restingstate at a small airway level−15×(inspiration+expiration) cycles withtidal volume average of 100 μl, or can be adjusted to accommodatedifferent breathings patterns and/or tidal volumes.

To visualize and measure the direction/rate of the gas flow or air flow,art-recognized techniques such as particle image velocimetry ormicron-resolution particle image velocimetry can be employed. Forexample, fluorescence beads can be added into the “airway lumen”channel, i.e., on top of the differentiated epithelial cells (e.g.,differentiated airway epithelial cells), and the movement of thefluorescent beads can be captured with a microscope. FIG. 14A is a setof snapshot images showing the movement of the fluorescent beads withinthe “airway lumen” channel of the device at a specific time point. Theleft panel is directed to a control device that did not receive airflowand shows partially polarized bead movements—i.e. some beads in onedirection, a few in the opposite direction. The right panel is directedto a device that received airflow for about 24 hrs and shows morepolarized bead movement towards the “mouth end.” This set-up can be, forexample, used to determine ciliary clearance rate of a particle. By wayof example only, FIG. 14B is a bar graph showing a higher ciliaryclearance rate of the fluorescent beads in the device that receivedairflow (breathing chip) than in the control device without airflow (thenon-breathing chip). Similarly, ciliary clearance rate of pathogens,compounds, and/or particulates introduced into the mesochannel can alsobe determined using the device described herein. In some embodiments,the pathogens, compounds, and/or particulates can be labeled with adetection molecule (e.g., a fluorescent molecule) for ease ofvisualization and/or tracking.

Co-Culture:

As used herein, the term “co-culture” refers to two or more differentcell types being cultured in a device described herein. The differentcell types can be cultured in the same channel (e.g., mesochannel ormicrochannel) and/or in different channels (e.g., one cell type in amesochannel and another cell type in a microchannel). For example, insome embodiments, in order to recapitulate in vivo microenvironment, insome embodiments, another side of the membrane 208 facing themicrochannel 250B can be cultured with blood vessel-associated cells,e.g., but not limited to, endothelial cells, fibroblasts, smooth musclecells, pericytes, or any combinations thereof. In one embodiment, asshown in FIG. 8, the side of the membrane 208 facing the microchannel250B is cultured with endothelial cells. As endothelial cells generallyplay a significant role in immune cell recruitment and/or extravasation,co-culture of tissue-specific epithelial cells (e.g., airway epithelialcells) on one surface of the membrane facing the mesochannel 250A withendothelial cells on another surface of the membrane facing themicrochannel 250B can create a physiologically-relevant model to performan immune cell recruitment assay, e.g., by introducing immune cells(e.g., but not limited to, CD8+ T cells, lymphocytes, monocytes,neutrophils) in the microchannel, followed by determination of thenumber of immune cells adhered onto the endothelial monolayer. In someembodiments, endothelial cells can also participate incytokine/chemokine secretion during a virus infection.

In some embodiments, the side of membrane 208 facing the microchannel250B can further comprise smooth muscle cells and/or fibroblasts. Whenthere is more than one cell type in a channel, a culture medium suppliedto the channel can comprise a mixture of culture media typically used toculture individual cell types.

In some embodiments, tumor cells can be co-cultured with normalepithelial cells in the mesochannel.

In some embodiments where the device models an intestine, the intestinalepithelial cells can be co-cultured with intestinal microbial flora inthe mesochannel.

In some embodiments, the device described herein can be used to createan in vitro model that mimics a tissue-specific condition. As usedherein, the term “tissue-specific condition” refers to any conditionthat can be diagnosed in a tissue of an organ in vivo. The condition canoccur naturally in the tissue in vivo (including, e.g., a normal healthycondition, or a condition induced or caused by a congenital defect), orinduced or caused by a condition-inducing agent or stimulant (e.g.,including, but not limited to an environmental agent). Examples of atissue-specific condition include, without limitations, a normal state,a disease-specific state, a pre-disease state, a disease remissionstate, a distressed state, an inflamed state, an infected state, and astimulated state. In these embodiments, the tissue-specific cells placedon the surface of the membrane facing the mesochannel can be adapted todisplay at least one characteristic associated with the tissue-specificcondition. For example, in some embodiments, patient- anddisease-specific epithelial cells and optional structural cells can becultured and differentiated on the surface of the membrane facing themesochannel, for example, to model chronic organ disorders such aschronic lung disorders, e.g., but not limited to chronic obstructivepulmonary disease (COPD), asthma, cystic fibrosis (CF), and fibroticconditions such as sarcoidosis, and idiopathic lung fibrosis,

In some embodiments, disease-specific cells can be obtained from one ormore patients diagnosed with the specific disease. For example,asthmatic, chronic obstructive pulmonary disease (COPD) and cysticfibrosis (CF)-associated airway cells can be obtained from one or moreasthmatic, COPD and CF patients, respectively.

In other embodiments, the tissue-specific cells (e.g., normaltissue-specific cells) can be contacted with a condition-inducing agentdescribed herein that is capable of inducing the tissue-specific cellsto acquire at least one characteristic associated with thetissue-specific condition. For example, lung infections can be modeledby introducing a biological and/or chemical agent, e.g., pathogens suchas influenza virus, and/or an immunostimulant (e.g.,polyinosinic:polycytidylic acid (usually abbreviated as poly I:C) tomodel lung infections, including bacterial and/or viral infections. Inone embodiment, cigarette smoke can be used to stimulate normal healthycells for inducing chronic obstructive pulmonary disease (COPD)phenotype. In another embodiment, asthmatic-like cells can be derivedfrom normal healthy cells by inducing inflammation in the normal healthycells, e.g., by exposure to a pro-inflammatory agent described herein.Pro-inflammatory agents are described below in the section “Additionalexamples of cytokines”. In some embodiments, the pro-inflammatory agentcan be TNF-alpha. In some embodiments, it can be desirable to induce anasthma-like phenotype in normal cells (rather than using diseased cellscollected from patients diagnosed with asthma), for example, to reduceor eliminate genetic variability/heterogeneity among different asthmaticdonors.

The stimulants or condition-inducing agents as described herein (e.g.,but not limited to, smoke particles, pathogens, cytokines such aspro-inflammatory agents, and/or drugs) can be delivered to the cells viadiffusion from the microchannel, and/or as an aerosol or liquid throughthe mesochannel. The aerosol of molecules or pathogens can be generatedon-chip, e.g., modifying the device described herein to integrate withan in vitro aerosol delivery device described in the PCT applicationserial nos. PCT/US12/37096 and PCT/US13/36569, the content of which areincorporated herein by reference. In one embodiment, as shown in FIG.21, an inertial impactor 2100 as described in the PCT application serialno. PCT/US12/37096 can be placed in the bottom portion 206 of the devicebody and fluidically connects to the mesochannel in the top portion 204of the device body. An access port 2102 can be placed on the lateralsurface of the bottom portion of the device body and fluidicallyconnects to the inertial impactor 2100. Thus, an aerosol produced froman aerosol-producing element can be introduced into the access port2012, flowing through the inertial impactor 2100 where larger dropletsof the aerosol are captured on the wall surface of the inertial impactor2100 (e.g., to prevent blocking of the mesochannel), while smallerdroplets of the aerosol continue to flow into the mesochannel.

As used herein, the term “immune cells” generally refer to restingand/or activated cells of the immune system involved in defending asubject against both infectious disease and foreign materials. Examplesof immune cells include, without limitations, white blood cellsincluding, e.g., neutrophils, eosinophils, basophils, lymphocytes (e.g.,B-cells, T-cells, and natural killer cells), monocytes, macrophages(including, e.g., resident macrophages, resting macrophages, andactivated macrophages); as well as Kupffer cells, histiocytes, dendriticcells, Langerhans cells, mast cells, microglia, and any combinationsthereof. In some embodiment, immune cells include derived immune cells,for example, immune cells derived from lymphoid stem cells and/ormyeloid stem cells.

In some embodiments, a tissue-specific condition, e.g., adisease-specific condition can be created by genetically modifyingnormal healthy cells, e.g., by silencing one or more genes, orover-expressing one or more genes. Methods of gene silencing include,but are not limited to, RNA interference (e.g., but not limited to smallinterfering RNA (siRNA), microRNA (miRNA), and/or short hairpin RNA(shRNA)), antisense oligonucleotides, ribozymes, triplex formingoligonucleotides, and the like. By way of example only, CF-associatedairway cells can be derived from normal healthy cells by a knock-out orsilencing of the cystic fibrosis transmembrane conductance regulator(CFTR) gene, in which the presence of at least one or more mutations isknown to cause CF. For example, a CFTR-targeting shRNA, siRNA, antisenseoligonucleotide, ribozyme, and/or triplex forming oligonucleotide can beintroduced into normal healthy airway cells (e.g., primary cells), e.g.,by a lentivirus system, in order to silence the CFTR gene, which can inturn result in a CF phenotype in the normal healthy cells.

In some embodiments, rhythm of airflow in the mesochannel of the devicedescribed herein can be adjusted, alone or in combination with a liquidmedium flowing in the microchannel, for example, to model acute lunginjuries—either physical or chemical, or with or without breathing,e.g., inhaled acids/alkali or ventilator-induced injuries.

In some embodiments where the devices described herein are used tocreate a disease-specific model, the devices can further comprise normalhealthy cells (e.g., obtained from one or more healthy donors) culturedin a separate central channel, e.g., to create a baseline forcomparison.

In some embodiments, the device can comprise both healthy anddisease-specific cells. In some embodiments, the device can include onlydisease-specific cells.

By way of example only, FIGS. 11A-11D illustrate the capability of usingone embodiment of the device described herein to model a bacterial/viralinfection. Upon differentiation of the airway epithelial cells intociliated and mucous-secreting (goblet) cells, the differentiated cellscan be challenged with pathogens (e.g., bacteria, fungus, and/or virus)and/or their associated stimuli (e.g., toll-like receptor 3 (TLR-3)ligand poly I:C, or pro-inflammatory agents, e.g., but not limited toTNF-α) in order to induce inflammation. A fluid comprising immune cellsdescribed herein (e.g., but not limited to, human monocytes) isintroduced into the “blood vessel” channel, either with a static fluidor a flowing fluid, to determine effects of a pro-inflammatoryagent-induced inflammation on cytokine/chemokine profiles of thedifferentiated cells and/or recruitment of immune cells described herein(e.g., but not limited to, monocytes and/or neutrophils). FIG. 11B showsthat TLR-3 activation (flu-like situation) stimulates release ofchemokines (e.g., monocyte chemoattractants and neutrophilchemoattractants) by the differentiated airway epithelial cells in thedevice. Cytokines or chemokines secreted into the fluid flowing in themesochannel and/or microchannel can be measured by collecting from theoutlet an aliquot of the fluid exiting the mesochannel and/ormicrochannel, which is then subjected to cytokine/chemokine expressionanalyses. FIG. 11C shows that TLR-3 stimulation enhances monocyteadhesion to differentiated epithelial cells. FIG. 11D shows thatdifferentiated epithelial cells after stimulation with a TLR-3 ligandpoly I:C significantly increases epithelial cells' gene expression ofIP-10.

In some embodiments, tumor cells can be co-cultured with tissue-specificepithelial cells on the surface of the membrane facing the mesochannel,e.g., to study metastasis of a tissue-specific cancer. In oneembodiment, lung cancer can be modeled by studying metastasis of tumorcells among the lung epithelial cells.

In some embodiments, a smoking lung-on-a-chip can be created byintroducing a flow of smoke particles across the mesochannel to studyeffect of smoke on function and transformation of airway and/or lungepithelial cells cultured on one surface of the membrane facing themesochannel, with or without endothelial cells lining another surface ofthe membrane facing the microchannel.

In some embodiments, the device described herein can be used to model atleast a portion of an intestine or gut and induce intestinal cells toundergo morphogenesis of three-dimensional (3D) intestinal villi. Forexample, human intestinal epithelial cells (e.g., epithelial cellsassociated with an intestine such as duodenum, jejunum, ileum, cecum,colon and an appendix) can be cultured on the surface of the membranefacing the mesochannel, with or without endothelial cells lining anothersurface of the membrane facing the microchannel. By exposing thecultured cells to a physiological peristalsis-motion produced bystretching and retracting the membrane (e.g., about 5% to about 20% at afrequency of about 0.05 Hz to about 0.3 Hz) and flowing a liquid at lowshear stress (e.g., 0.02 dyne cm⁻²) in the mesochannel, the intestinalcells can grow into folds and form tubular projections (villi)projecting into the mesochannel (which is modeled as “intestinal lumen”)to recapitulate the 3D structure. Formation of these intestinalvilli-like structures can provide increased surface area that mimics theabsorptive efficiency of human intestine, and/or enhanced cytochromeP450 3A4 isoform-based drug metabolizing activity (Kim et al., 2012 LabChip, 12: 2165-2174 and Kim et al., 2013 Integrative Biology, firstpublished online 26 Jun. 2013; DOI: 10.1039/C3IB40126J). Thesefunctional features of human intestine recapitulated in a controlledmicrofluidic environment can be used for transport, absorption, andtoxicity studies, drug testing as well as development of intestinaldisease models and screening for therapeutic agents. Examples ofintestinal diseases that can be modeled using the devices describedherein include, but are not limited to, inflammatory bowel disease,Crohn's disease, ulcerative colitis, celiac disease, angiodysplasia,appendicitis, bowel twist, chronic functional abdominal pain, coeliacdisease, colorectal cancer, diverticular disease, endometriosis,enteroviruses, gastroenteritis, Hirschsprung's disease, ileitis,irritable bowel syndrome, polyp, pseudomembranous colitis, or anycombinations thereof.

Drugs intended for oral administration generally require goodbioavailability in order to achieve therapeutic concentrations at thetargeted site of action. Good bioavailability implies that an effectiveamount of drug is able to reach the systemic circulation. However, drugabsorption via oral route can be affected by drug properties and/or thephysiology of the gastrointestinal tract, including drug dissolutionfrom the dosage form, the manner in which drug interacts with theaqueous environment and membrane, permeation across membrane, andirreversible removal by first-pass organs such as the intestine, liver,and lung (Martinez and Amidon, 2002 J Clin Pharmacol 42: 620-643). Inparticular, the majority of drug absorption generally occurs at thesmall intestine where the presence of villi and microvilli markedlyincreases the absorptive area. Thus, in some embodiments, the devicesmodeling the function of an intestinal villus structure as describedabove can be used to assess intestinal absorption, metabolism, and/orexcretion of a test agent for the prediction of its bioavailability. Insome embodiments, the devices modeling the function of the intestinalvillus structure can be fluidically connected to another devicemimicking a target tissue to be treated by the test agent.

In some embodiments, the devices described herein can be used todetermine an effect of a test agent on the cells on one or both surfaceof the membrane. Effects of a test agent can include, but are notlimited to, ciliary clearance, villi absorption, cell viability,permeability of a cell layer, cell morphology, protein expression, geneexpression, cell adhesion, adhesiveness of immune cells, celldifferentiation, cytokine or chemokine production, inflammation, or anycombinations thereof.

In accordance with some embodiments of the invention, the devicesdescribed herein can be used to determine an efficacy of a test agentupon exposure of the cells on one or both surfaces of the membrane tothe test agent. For example, the efficacy of a test agent can bedetermined by measuring response of the cells and/or at least onecomponent present in a fluid (e.g., gaseous and/or liquid fluid) withinthe device or present in an output fluid (e.g., gaseous and/or liquidfluid) from the device after exposure to the test agent. As used herein,the term “efficacy” generally refers to ability of a test agent toproduce a desired effect or outcome. Depending on the nature and/or typeof the test agents, examples of desired effects or outcomes include, butare not limited to, therapeutic effect, cytotoxicity, cell growth, celldifferentiation, improved or reduced cell function or phenotype (e.g.,but not limited to, ciliary clearance, permeability of a cell layer,cell migration, expression and/or secretion of a protein or cytokinethat can be affected by cell exposure to the test agent), and anycombinations thereof. The term “therapeutic effect” as used hereinrefers to a consequence of treatment, the results of which are judged tobe desirable and beneficial.

In accordance with some embodiments of the invention, the devicesdescribed herein can be used to determine toxicity of a test agent uponexposure of the cells on one or both surfaces of the membrane to thetest agent. For example, the toxicity of a test agent can be determinedby measuring response of the cells and/or at least one component presentin a fluid (e.g., gaseous and/or liquid fluid) within the device orpresent in an output fluid (e.g., gaseous and/or liquid fluid) from thedevice after exposure to the test agent. As used herein, the term“toxicity” refers to ability of a test agent to induce or cause anyadverse and/or side effect on a cell and/or even cell death. Forexample, the toxicity of a test agent can be characterized by itsability to induce or cause an adverse effect on cell function and/orphenotype, including, but not limited to, alteration in cell metabolism,mutagenicity, carcinogenicity, teratogenicity, DNA damage, protein ormembrane damage, cell energy depletion, mitochondrial damage,genotoxicity, apoptosis, cell death, cell rupture, and any combinationsthereof.

In accordance with some embodiments of the invention, the devicesdescribed herein can be used to determine a mechanism of action uponexposure of the cells on one or both surfaces of the membrane to thetest agent. For example, the mechanism of action can be determined bymeasuring response of the cells and/or at least one or more componentspresent in a fluid (e.g., gaseous and/or liquid fluid) within the deviceor present in an output fluid (e.g., gaseous and/or liquid fluid) fromthe device after exposure to the test agent. As used herein, the term“mechanism of action” refers generally to a cellular pathway orbiological interaction through which an agent exerts its biologicaleffect on a cell. For example, when an agent is a drug substance,mechanism of action can refer to the biochemical interaction throughwhich a drug substance produces its pharmacological effect. Depending onthe nature and/or type of test agents, the mechanism of action can beassociated with any art-recognized cellular pathways or biologicalinteraction, e.g., including, but not limited to, protein synthesis,cell migration, chromatin regulation/epigenetics or acetylation, MAPKsignaling, apoptosis, autophagy, PI3K/Akt signaling, translationcontrol, cell cycle/checkpoint, Jak/Stat Pathway, NF-B signaling,TGF-/Smad signaling, lymphocyte signaling, angiogenesis, cytoskeletalsignaling, cell adhesion, cell metabolism, cell development and/ordifferentiation, tyrosine kinase/adaptors, protein stability, proteinfolding, nuclear receptor signaling, and any combinations thereof.Accordingly, in some embodiments, a mechanism of action can encompass amechanism of efficacy and/or toxicity of a test agent.

In accordance with some embodiments of the invention, thetissue-specific epithelial cells on the surface of the membrane facingthe mesochannel can be contacted with a test agent. The test agent canbe delivered to the cells as an aerosol or liquid through themesochannel or “airway lumen” channel and/or via diffusion from themicrochannel or “blood vessel” channel. As described earlier, an aerosol(e.g., of the test agent) can be generated on-chip, e.g., modifying thedevice described herein to integrate with an in vitro aerosol deliverydevice described in the PCT application serial nos. PCT/US12/37096 andPCT/US13/36569, the content of which are incorporated herein byreference.

Any test agent can be introduced into the device described herein todetermine its effect on the cells. Examples of the test agent caninclude, but are not limited to, proteins, peptides, antigens,nanoparticles, environmental toxins or pollutant, cigarette smoke,chemicals or particles used in cosmetic products, small molecules, drugsor drug candidates, vaccine or vaccine candidates, aerosols,inflammatory molecules, naturally occurring particles including pollen,chemical weapons, single or double-stranded nucleic acids, viruses,bacteria and unicellular organisms.

Effects of the test agent on the cells can be determined by measuringresponse of the cells on at least one side of the membrane to the testagent, the gaseous fluid exiting the first sub-channel, the liquid fluidexiting the second sub-channel, or any combinations thereof; andcomparing the measured response with the cells not contacted with thetest agent. Various methods to measure cell response are known in theart, including, but not limited to, cell labeling, immunostaining,optical or microscopic imaging (e.g., immunofluorescence microscopyand/or scanning electron microscopy), spectroscopy, gene expressionanalysis, cytokine/chemokine secretion analysis, metabolite analysis,polymerase chain reaction (PCR), immunoassays, ELISA, gene arrays,spectroscopy, immunostaining, electrochemical detection, polynucleotidedetection, fluorescence anisotropy, fluorescence resonance energytransfer, electron transfer, enzyme assay, magnetism, electricalconductivity (e.g., trans-epithelial electrical resistance (TEER)),isoelectric focusing, chromatography, immunoprecipitation,immunoseparation, aptamer binding, filtration, electrophoresis, use of aCCD camera, mass spectroscopy, or any combination thereof. Detection,such as cell detection, can be carried out using light microscopy withphase contrast imaging and/or fluorescence microscopy based on thecharacteristic size, shape and refractile characteristics of specificcell types. Greater specificity can be obtained using optical imagingwith fluorescent or cytochemical stains that are specific for individualcell types or microbes.

In some embodiments, adhesion of immune cells that are introducedthrough the “blood vessel” channel to the endothelium or membrane can bemeasured to determine effects of a test agent on immune response.

In some embodiments where the tissue-specific cells to be assayed areadapted to be condition-specific (e.g., disease-specific), exposure ofthe tissue-specific cells to a test agent followed by determination ofthe effect of the test agent on the cells can facilitate identificationof a therapeutic agent for treatment of the condition. For example,FIGS. 12A-12D illustrate an exemplary method of evaluating an effect ofdifferent agents on differentiated airway epithelial cells andoptionally immune cells during an infection in a device in accordancewith an embodiment, and experimental data resulting therefrom. FIG. 12Ais a schematic diagram illustrating an example method to evaluate aneffect of different agents during an infection simulated in the device.Primary human epithelial cells from chronic obstructive pulmonarydisease (COPD) patients are seeded on the membrane in the mesochannel(an “airway lumen” channel) for differentiation into ciliated and/ormucus-secreting cells following the differentiation method as describedin FIG. 5A. Upon differentiation of the COPD epithelial cells, anothersurface of the membrane (facing the microchannel, the “blood vessel”channel) can be seeded with or without endothelial cells. The cells inthe device can be optionally starved using basal medium, followed bytreatment with different test agents (e.g., DMSO as a control,budesonide, and BRD4 inhibitor compounds 1 and 2 obtained from apharmaceutical company). The agents can be delivered to thedifferentiated epithelial cells via diffusion from the “blood vessel”channel. The pre-treated differentiated COPD epithelial cells are thenchallenged with TLR-3 ligand poly I:C (e.g., about 10 μg/mL delivered asan aerosol or liquid flowing in the mesochannel) to stimulate TLR-3 andmimic viral infection. Secreted cytokines and chemokines from thedifferentiated COPD epithelial cells can be quantified in theflow-through of the “blood vessel” channel and/or from the apical washof the “airway lumen” channel. In some embodiments, a fluid comprisingimmune cells (e.g., human monocytes) can be introduced into the “bloodvessel” channel, either with a static fluid or a flowing fluid, todetermine effects of TLR-3-induced inflammation on recruitment of immunecells (e.g., monocytes and/or neutrophils). FIG. 12B shows production ofrepresentative cytokines and chemokines (e.g., monocyte chemoattractantsand neutrophil chemoattractants) by the differentiated COPD epithelialcells (pretreated with different agents prior to exposure to a TLR-3ligand poly I:C) and released into the “blood vessel” channel, andindicates that compound 2 is more potent than compound 1 in reducingcytokine/chemokine secretion in response to the simulated viralinfection. In addition, FIG. 12F shows that compound 2 is more potent inreducing neutrophil adhesion, whereas compound 1 did not have sucheffect, and such result is consistent with and validates thepharmaceutical company's in-house data on potency of compound 2 inreducing inflammation. Thus, the devices and methods described hereincan be used to screen drugs.

In some embodiments where the tissue-specific cells arepatient-specific, exposure of the patient-specific cells to a testagent, followed by determination of the effect of the test agent on thecells can facilitate identification of a personalized treatment for asubject.

In some embodiments where the tissue-specific cells are patientpopulation-specific, exposure of the patient population-specific cellsto a test agent, followed by determination of the effect of the testagent on the cells can facilitate identification of a treatmentspecified for that particular patient population. As used herein, theterm “patient population-specific” refers to cells collected from apopulation of patients sharing at least one or more phenotypes and/orcharacteristics (e.g., but not limited to, specific gene mutation,ethnicity, gender, life styles, BMI, resistance to treatment, and anycombinations thereof) other than the disease or disorder.

In some embodiments, one or more devices described herein can be used incombination with a pharmacokinetic (PK) model, a pharmacodynamic (PD)model, or a PK-PD model to quantitatively analyze the effect of an agentto be tested. For example, a series of devices, each modeling a tissue,e.g., one for gut, one for liver, and another one for heart, can beconnected to provide a microphysiological system that can be used todetermine the fate of an agent administered into the system. The term“pharmacokinetics” is used herein in accordance with the art, and refersto the study of the action of agents, e.g., drugs, in the body, forexample, the effect and duration of drug action, the rate at which theyare absorbed, distributed, metabolized, and eliminated by the body etc.(e.g. the study of a concentration of an agent, e.g., a drug, in theserum of a patient following its administration via a specific dose ortherapeutic regimen). The term “pharmacodynamics” is used in accordancewith the art, and refers to the study of the biochemical andphysiological effects of an agent, e.g., a drug, on a subject's body oron microorganisms such as viruses within or on the body, and themechanisms of drug action and the relationship between drugconcentration and effect (e.g. the study of a pathogen, e.g., a virus,present in a patient's plasma following one or more therapeuticregimens). Methods for PK-PD modeling and analysis are known in the art.See, e.g., Bonate, P. L. (2006). Pharmacokinetic-PharmacodynamicModeling and Simulation. New York, Springer Science & Business Media;Gabrielsson, J. and D. Weiner (2000); and Pharmacokinetic andPharmacodynamic Data Analysis: Concepts and Applications. Stockholm,Swedish Pharmaceutical Press. For example, a PK model can be developedto model a microphysiological system comprising a plurality of thedevices described herein, wherein each device can model a differenttissue that can produce an effect (e.g., absorption, metabolism,distribution and/or excretion) on an agent to be administered. Toconstruct a PK model for a device described herein, mass balanceequations describing the flow in, flow out, and metabolism of an agentcan be set up for each mesochannel and microchannel. A PD model can beintegrated into each device described herein, describing the kinetics ofpotential cell response (e.g., inflammation, cytokine release, ligandbinding, cell membrane disruption, cell mutation and/or cell death) ineach device that mimics a tissue or an organ. This in vitro/in silicosystem, combining one or more devices described herein with anintegrated PK-PD modeling approach, can be used to predict drug toxicityin a more realistic manner than conventional in vitro systems. In someembodiments, one or more of the devices described herein can be used toquantify, estimate or gauge one or more physical-chemical,pharmacokinetic and/or pharmacodynamic parameters. Variousphysical-chemical, pharmacokinetic and pharmacodynamic parameters areknown in the art, including, for example, the ones discussed in theaforementioned references. Exemplary physical-chemical, pharmacokineticand pharmacodynamic parameters include, but are not limited to,permeability, log P, log D, volume of distribution, clearances(including intrinsic clearances), absorption rates, rates of metabolism,exchange rates, distribution rates and properties, excretion rates,IC50, binding coefficients, etc.

In some embodiments, the devices described herein can be used for targetidentification/validation. For example, the devices described herein canbe used to mimic a tissue-specific condition as described herein (e.g.,a disease or disorder) in order to elucidate the molecular mechanismunderlying a disease or a condition, the identification of candidatetarget molecules and the evaluation of said target molecules. In someembodiments, use of genetically modified cells, e.g., by silencing orover-expressing a specific gene, in the devices described herein can beused to identify target molecules for a specific disease. Once such avalidated target molecule, e.g., ligand, receptor, transcription factor,and/or enzyme, which is herein referred to also as target, isidentified, drug candidates directed to the target (e.g., suppression oractivation) can be tested. The drug candidate can be introduced to thedisease-specific cells in the devices described herein and cell responseto the drug candidate can be measured to validate the identified target.This can also promote drug discovery for a specific disease orcondition. In many cases such drug candidates can be members of acompound library which can comprise synthetic and/or natural compounds.Combinatorial libraries can also be used.

Similarly, the devices described herein can be used to mimic aphysiological environment under which a drug fails during a clinicaltrial. Thus, mechanism of action of the drug can be studied tofacilitate identification of a new drug target.

In some embodiments, the devices described herein can be cultured withanimal cells (e.g., but not limited to, pig cells, rabbit cells, dogcells, mouse cells, and/or rat cells) to determine response of theanimal cells to an agent introduced into the devices described herein.The measured response of the animal cells in the devices can then becorrelated with the actual response occurred in vivo when the agent isadministered to a living animal (e.g., a pig, a rabbit, a dog, a mouse,and/or a rat). By identifying the correlation between the in vitro andin vivo responses in one or more animal models, one can extrapolate orpredict effect of the agent on a human subject in vivo, based on themeasured responses of the human cells to the agent in the devices.Additionally or alternatively, a therapeutic dose of an agent for ahuman subject can be determined accordingly.

In some embodiments, the combination of simulated breathing through the“airway lumen” channel and ability to connect to two or more devicesdescribed herein (e.g., in series and/or in parallel) can allow studyinghow airborne pathogens, e.g., but not limited to virus, bacteria,respiratory syncytial virus, influenza virus, or MycobacteriumTuberculosis (MTB), from a “pathogen-infected” device can infect one ormore “non-infected” devices, as shown in FIG. 15. In these embodiments,a first device comprising pathogen-infected epithelial cells can beadapted to connect, e.g., in series and/or in parallel, to at least onea second device comprising non-infected cells. The distance between twodevices can be adjusted to simulate closeness of contact between twosubjects and/or control the rate of airborne pathogen transmissionbetween two subjects.

In some embodiments, the pathogen-infected epithelial cells can beobtained from one or more infected subjects. In some embodiments, thenon-infected cells can be obtained from one or more normal healthysubjects and/or subjects with a disease or disorder such as arespiratory disease. An air flow can then be directed from the “airwaylumen” mesochannel of the first device to the “airway lumen” mesochannelof the second device. Response of the non-infected cells (includingimmune cells) upon exposure to the air flow from the first device aswell as response of the infected cells (including immune cells) can bemeasured to determine transmissibility of airborne pathogens.

In some embodiments, the “airborne pathogen transmission” model asdescribed above can be used to assess infectivity or virulence of apathogenic strain such as a strain of virus. For example, at least twoor more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) “non-infected”devices can be connected in series and/or in parallel. In each“non-infected” device, one surface of the membrane facing themesochannel can comprise lung-associated cells (e.g., lung cells, nasalcells, tracheal cells, airway cells, and/or bronchial cells) while theother surface of the membrane can comprise blood vessel-associated cellsor not. Then, a pathogenic strain to be assessed can be introduced intothe mesochannel of one of the connected devices. Recruitment and/orinfiltration of immune cells can be measured in each device to determineinfectivity or virulence of the introduced pathogenic strain. Typically,a viral infection can induce an immune response, which can include,e.g., increased immune cell recruitment and/or infiltration.

The lung-associated or other appropriate tissue-specific cells to beinfected in each “non-infected” device can be collected from a differentsubject or subject population having distinct phenotypes (e.g., by age,sex, genotypes, life-style such as smoking, frequent exercises, anddiets, diseases or disorders). For example, children and elderly aregenerally more prone to a viral infection, and subjects with arespiratory disease such as asthmatic patients can suffer from viralexacerbation of the respiratory disease when they are exposed to avirus. Accordingly, by measuring the response of immune cells fromdifferent subject populations in the individual connected devices, onecan also identify risk populations for a pathogenic strain.

In some embodiments, the “airborne pathogen transmission” model asdescribed herein can be used to assess risk of a novel (i.e., new inhumans) virus strain acquiring the ability to spread easily andefficiently in humans. Ten evaluation criteria that Centers for DiseaseControl and Prevention (CDC) currently use to measure the potentialpandemic risk posed by influenza A viruses (Influenza Risk AssessmentTool accessible atwww.cdc.gov/flu/pandemic-resources/tools/risk-assessment.htm) can beused as guidelines to determine the potential pandemic risk associatedwith emergence of a novel virus strain using the devices describedherein. For example, a novel influenza virus can be introduced to“non-infected human” devices comprising human cells to determine ifhuman-to-human transmission can occur and/or how frequently and easilythe transmission can occur after a direct and prolonged “contact” orconnection between the devices. In addition or alternatively, a novelinfluenza virus can be introduced to “non-infected animal” devicescomprising various animal cells to determine what kind of animals can beimpacted by the influenza virus, because the likelihood of human contactwith some animals can be higher (e.g., domestic birds vs. wild birds),which can influence the pandemic risk. Additionally or alternatively, anovel influenza virus can be introduced to “non-infected” devicescomprising different tissue-specific cells to determine the types oftissues and/or cells the virus is more prone or susceptible to infection(e.g., nose tissue and cells vs. deep lung tissue and cells).

In some embodiments, the “airborne pathogen transmission” model asdescribed above can also be used to determine prophylactic ortherapeutic efficacy of an anti-pathogen agent (e.g., anti-viral agent)or a vaccine against an airborne pathogen. For example, for prophylacticagents or vaccines, the normal healthy cells can be pre-exposed to anagent or vaccine of interest and then exposed to an airflow contaminatedwith the airborne pathogens from the first device. By measuring theresponse of the non-infected cells and optional immune cells to theairborne pathogens, efficacy (e.g., immunogenicity) and/or safety of theagent or vaccine can be determined. Similarly, for therapeutic agents orvaccines (e.g., anti-viral vaccines), the pathogen-infected cells in thefirst device can be treated with an agent or vaccine of interest beforedirecting an air flow from the first device to the second devicecomprising non-infected cells. A reduction or an inhibition of thetransmissibility of the airborne pathogens is indicative of the efficacyof a therapeutic agent or vaccine.

Additional examples of using one or more embodiments of the devicesdescribed herein for development of a vaccine, e.g., a mucosal vaccine,are described in detail below.

Uses of the devices described herein to determine transmissibility ofairborne pathogens, identify risk populations for airborne pathogensand/or to develop agents or vaccines against airborne pathogens areprovided herein as illustrative examples. As one of skill in the artwill appreciate, the “airborne pathogen transmission” model describedherein can be readily adapted to mimic transmission of a bodyfluid-borne pathogen such as hepatitis B, hepatitis C, and/or HIV/AIDSbetween subjects. For example, a droplet of a liquid fluid from an“infected” device can be introduced to a liquid fluid in a“non-infected” device. Response of the non-infected cells (includingimmune cells) upon exposure to the infected liquid fluid as well asresponse of the infected cells (including immune cells) can be measuredto determine transmissibility of body fluid-borne pathogens, e.g.,pathogens that can be transmitted through blood, semen, vaginalsecretions, cerebrospinal fluid, synovial fluid, pleural fluid,pericardial fluid, peritoneal fluid, amniotic fluid, saliva, or anycombinations thereof.

In some embodiments, the exclusion of fluorescently labeled largemolecules (e.g. dextrans of different weight or FITCs) can bequantitated to determine the permeability of the membrane and thusassess the barrier function of the epithelium, e.g., in atissue-specific condition (e.g., but not limited to, COPD, asthma, andsmoking). For example, flowing a fluid containing fluorescently labeledlarge molecules (e.g., but not limited to, inulin-FITC) into amesochannel cultured with differentiated epithelium can provide anon-invasive barrier measurement. As a functional tight junction barrierwill prevent large molecules from passing through the epithelium fromthe mesochannel to the microchannel, the absence of the detection of thefluorescently labeled large molecules in the microchannel is indicativeof a functional barrier function of the epithelium.

Additionally, histological, biochemical, microfluorimetric and/orfunctional techniques can be employed to demonstrate formation of afunctional airway-endothelial that reproduces the key structuralorganization of its in vivo counterpart on the membrane 208.

In an example, the gas exchange function of the tissue-tissue interfaceself assembled on membrane 208 can be determined by injecting differentfluids, each having their own oxygen partial pressures and blood, intothe respective mesochannel and microchannel 250A, 250B, whereby themesochannel 250A acts as the “airway lumen” compartment and themicrochannel 250B acts as the “microvascular” or “blood vessel”compartment. A blood-gas measurement device preferably within the device200 is used to measure the level of oxygen in the blood in therespective sections 250A, 250B before and after the passing of the bloodthrough the device. For example, blood can flow through the channel 250Bwhile air is being injected into the upper channel 250A, whereby theexiting air is collected and measured to determine the oxygen levelusing an oximeter. Oximeters can be integrated with the existing systemor as a separate unit connected to the outlet port of one or morecentral sub-channels. In an embodiment, air or another medium withaerosols containing drugs or particulates can flow through the device,whereby the transport of these drugs or particulates to the fluidflowing in the “microvascular” microchannel (e.g., blood, culturemedium) via the membrane is then measured. In some embodiments,pathogens or cytokines can be added to the air or gaseous medium sideand then the adhesion of immune cells introduced in the microvascularmicrochannel to nearby capillary endothelium and their passage alongwith edema fluid from the blood side to the airway side, as well aspathogen entry into blood, can be measured.

Since the functionality of an epithelium requires polarization ofconstituent cells, the structure of the membrane can be visualized usingtransmission electron microscopy, immunohistocytochemistry, confocalmicroscopy, or other appropriate means to monitor the polarization ofthe airway epithelial cell side of the membrane 208. In an airway mimicembodiment, a florescent dye can be applied to the mesochannel andmicrochannel 250A, 250B to determine pulmonary surfactant production bythe airway epithelium at the membrane 208. In particular, airwayepithelial cells on the membrane 208 can be monitored by measuring thefluorescence resulting from cellular uptake of the fluorescence dye thatspecifically labels intracellular storage of pulmonary surfactant (e.g.quinacrine) or using specific antibodies.

One of the unique capabilities of the device 200 allows development ofin vitro models that simulate inflammatory responses of the airway orbronchus at the organ or tissue level to allow study of how immune cellsmigrate from the blood, through the endothelium and into the airwaycompartment. One way this is achieved can be by controlled andprogrammable microfluidic delivery of pro-inflammatory agents describedherein (e.g. but not limited to, IL-1β, TNF-α, IL-8, silica micro- andnanoparticles, pathogens) to the differentiated airway epithelial cellsin the mesochannel 250A as well as whole human blood flowing or culturemedium containing circulating or static immune cells described herein(e.g., white blood cells such as neutrophils, and monocytes) in themicrochannel 250B. Electrical resistance and short circuit currentacross the membrane can be monitored to study changes in the vascularpermeability, extravasation of fluid and cell passage into the airwayspace during inflammatory responses. Microscopy imaging, e.g.,fluorescence microscopy, can be used to visualize dynamic cell motilebehavior during the extravasation response.

In some embodiments, the device described herein can be used to developa mucosal immunity platform, e.g., to study immune cell recruitment,maturation, and activation, cell killing, and drainage (e.g., as shownin FIG. 16). Mucosal immunity is a form of protective immunity that actsat mucosal surfaces of the gastrointestinal and/or respiratory tracts toprevent colonization by ingested and inhaled microbes. There aremucosa-associated lymphoid tissues (MALT), such as tonsils and Peyer'sPatches, that act to prevent infection. The mucosal layers are usuallylined or protected by epithelial barriers. This layer of epitheliumserves as the first line of defense against microbes. If microbes breachthe epithelial layer, mucosal tissues are the sites of immunologicalactivity. When epithelial cells detect presence of dangerous microbialcomponents such as pathogen-associated molecular patterns, they sendcytokine and chemokine signals to underlying mucosal cells such asmacrophages and dendritic cells to trigger an immune response.Epithelial cells are able to regulate these responses so thatundesirable responses are not activated by normal flora that could leadto mucosal inflammation. Accordingly, in some embodiments, to develop amucosal immunity model, the surface of the membrane facing themesochannel can be coated with gastrointestinal or respiratoryepithelial cells to mimic a portion of a gastrointestinal or respiratorytract, while another surface of the membrane facing the microchannel canbe coated with mucosal cells or immune cells such as macrophages anddendritic cells.

In some embodiments, the mucosal immunity model can be used to develop amucosal vaccine (e.g., a mucosal vaccine to Strep) and/or optimize avaccine dosage. The regulation of the epithelial layer has beenpresenting a challenge for mucosal vaccine dosage. For example, if theconcentration of a mucosal vaccine is not high enough, the mucosalimmunity will not recognize it as a threat and no immunity woulddevelop. Finding the correct concentration has been a challenge and beendifficult to measure because the vaccine can be diluted in mucosalsecretions, captured in the mucus, attacked by proteases and nucleases,and can be excluded by epithelial barriers. Using the mucosal immunitymodel developed in one or more embodiments of the device describedherein, epithelial cells or differentiated epithelial cells can bepre-exposed to different vaccine test candidates and/or various dosagesof the same, and then challenged with a microbe against which issupposed to be vaccinated. By measuring the response of the epithelialcells and/or immune cells to the microbes, efficacy (e.g.,immunogenicity), safety and/or optimum dosage of the vaccine testcandidates can be determined.

Depending on the administration routes of a vaccine, e.g., but notlimited to, intranasal, oral, intramuscular, subcutaneous, orintradermal, the membrane of the device described herein can be coatedwith different cell types to mimic the microenvironment where thevaccine exerts an effect. For example, as described above, the membranecan be coated with respiratory epithelial cells for development ofintranasal vaccines, or gastrointestinal epithelial cells for oralvaccines.

As discussed above, in some embodiments, the devices described hereincan be used to model an infectious disease, to determinetransmissibility of an infectious pathogen, and/or to identify effectiveagents (e.g., drugs molecules, and/or vaccine) for therapeutic and/orprophylactic treatments. Various methods can be used to detect thepresence or absence of infection in the devices described herein. Forexample, where fluorescently-labeled (e.g., GFP-expressing) pathogens(e.g., virus or bacteria) are used, normal healthy cells that areinfected with the fluorescently-labeled pathogens can be directlyfollowed over time or real-time by fluorescent microscopy. Alternativelyor additionally, the infection-suspected cells can be immuno-stained forviral/bacterial proteins and detected by immunofluorescence. In someembodiments where virus or bacteria can produce a cytopathic effect oninfected cells, e.g., causing damages to the infected cells' epithelium,the integrity of the infection-suspected cells' epithelium can beexamined over time under light or fluorescent microscopy.

Additional methods that can be used to detect the presence or absence ofinfection in the device described herein can include, e.g., but are notlimited to, quantification of pathogen (e.g., virus) replication, which,for example, can be measured by collecting effluent ofinfection-suspected cells from the mesochannel (termed “apical wash”,e.g., using cell culture medium) and/or effluent from the microchannel(termed “basal medium”) and then titrating pathogen growth over time inthe apical wash and/or basal medium using a plaque assay. Alternativelyor additionally, cytokines/chemokines secreted by theinfection-suspected cells can be determined by analysis of effluentscollected from the mesochannel and/or the microchannel. Somecytokines/chemokines such as CXCL10 or IL-8 can be significantlyelevated in the device with the infected cells as compared tonon-infected cells. In some embodiments where cellular antiviralproteins such as MX proteins can be up-regulated following infection ofthe cultures, the cellular antiviral proteins such as MX proteins can bestained in the infection-suspected cells for immunofluorescencedetection. In some embodiments, expression analysis of at least one ormore genes that are known to be upregulated following pathogen (e.g.,viral/bacterial) infection (as compared to non-infected cells) can beperformed on the infection-suspected cells, e.g., by microarray and/orquantitative real-time polymerase chain reaction (qRT-PCR).

Without wishing to be limiting, in other embodiments, the device 200 canalso be used to examine how nanomaterials or particulates behave withrespect to the airway-tissue interface. In particular, nanomaterials(e.g. silica nanoparticles, superparamagnetic nanoparticles, goldnanoparticles, single-walled carbon nanotubes) can be applied to theairway surface of the membrane 208 to investigate potential toxiceffects of nanomaterials on airway or endothelial cells grown on themembrane 208, as well as their passage from the airway channel into theblood channel. For instance, sensors 120 can be used to monitortransmigration of nanomaterials through tissue barriers formed on themembrane 208 and nanomaterial-induced changes in barrier functions suchas gas exchange and fluid/ion transport.

The device 200 permits direct analysis of a variety of important areasof airway/bronchial biology and physiology including but not limited togas exchange, fluid/ion transport, inflammation response, infection(e.g., viral or bacterial infection), edema/respiratory distresssyndrome, cancer and metastasis development, fungal infection, ciliaryclearance of particulates, epithelial differentiation, cytokineproduction, drug delivery as well as drug screening, biodetection, andpulmonary mechanotransduction. In addition, the device 200 allows foraccurately modeling biological tissue-tissue interfaces found in otherphysiological systems that require taller channel height to supportoptimal cell culture, form a stratified structure, and/or reduce shearon the cells, including, but not limited to, skin, liver, gut, heart,intestine, choroid plexus, gastrointestinal tract, glomerulus, andcancerous tumor microenvironment. As stated above, more than one device200 can be multiplexed and automated to provide high-throughput analysisof cell and tissue responses to drugs, chemicals, particulates, toxins,pathogens or other environmental stimuli for drug, toxin and vaccinescreening, as well as toxicology and biodetection applications. Thedevice can be used for studying complex tissue and organ physiology invitro, as well as tissue and organ engineering in vivo withbiocompatible or biodegradable devices.

In an embodiment, the device 200 can be used to produce artificialtissue layers therein. In the embodiment, two or more different types ofcells are applied on opposing surfaces of the membrane 208 and grownunder conditions that mimic the appropriate physiological environments.Additionally or alternatively, a pressure differential can be appliedbetween the central channel and at least one of the operating channelswhich causes the channel walls to move and thus causes the membrane 208to undergo expansion/contraction along its plane.

To further demonstrate the device's capabilities to reconstitute theintegrated organ-level responses in the airway, a more sophisticatedmodel can be developed that incorporates circulating or staticblood-borne immune cells and reproduced the key steps of airwayinflammation. Generally, inflammatory responses in the airway involve ahighly coordinated multistep cascade of epithelial production andrelease of early response cytokines, activation of vascular endotheliumthrough upregulation of leukocyte adhesion molecules and subsequentleukocyte infiltration from the pulmonary microcirculation into theairway space. To simulate this process, the apical surface of the airwayepithelium can be first stimulated, e.g., with tumor necrosis factor-α(TNF-α), which is a potent pro-inflammatory mediator, and endothelialactivation can be examined, e.g., by measuring the expression ofintercellular adhesion molecule-1 (ICAM-1). In response to TNF-αstimulation of the airway tissue, the endothelial cells on the oppositeside of the membrane can generally increase their surface expression ofICAM-1. Furthermore, the activated endothelium can support capture andfirm adhesion of human neutrophils flowing in the vascular microchannel,which did not adhere in the absence of cytokine exposure. Treatment ofthe epithelial cells with low doses of TNF-α can result in weakactivation of the endothelium, which caused captured neutrophils to rollcontinuously in the direction of flow without being arrested. Thetransmigrated neutrophils then emigrate onto the apical surface of theairway epithelium preferentially through paracellular junctions and areretained on the epithelial layer in spite of fluid flow and cyclicstretching. These sequential events can replicate the entire process ofneutrophil recruitment from the microvasculature to the airwaycompartment, which is a hallmark of airway inflammation.

In another example, the device 200 utilizes the porous membrane 208,whereby airway or bronchial epithelial cells are grown on one side ofthe membrane 208 facing the mesochannel 250A and endothelial cells,fibroblasts, smooth muscle cells, and/or pericytes are maintained on theother side of the membrane 208 facing the microchannel 250B. During theoperation of the device 200, these two cells layers communicate witheach other through passage of chemical and molecular cues through thepores on the membrane 208. This communication can be monitored andanalyzed to understand how the cells function differently as atissue-tissue interface, with or without physiological mechanicalsimulation, and compared to when grown as single tissue types inisolation as in standard tissue culture systems. By monitoring changesin cell and tissue physiology, as well as passage of chemicals,molecules, particulates and cells across this tissue-tissue interface,information is obtained which can be used to produce more effectivedrugs or therapies, to identify previously unknown toxicities, and tosignificantly shorten the timescale of these development processes. Inparticular, the behavior of cells in such a controlled environmentallows researchers to study a variety of physiological phenomena takingplace in the systems mentioned above that can not be recreated usingconventional in vitro culture techniques. In other words, the device 200functions to create a monitorable artificial blood or liquid-air barrieroutside a patient's body and in a controllable environment that stillretains key physiological functions and structures of the airway orbronchus. It should be noted that although the device above is describedin terms of mimicking airway or bronchus function, the device can easilybe configured to mimic other physiological systems such as peristalsisand absorption in the gastrointestinal tract containing living microbialpopulations, perfusion and urine production in the kidney, function ofthe blood-brain barrier, effects of mechanical deformation on skinaging, bone marrow-microvessel interface with hematopoietic stem cellniche, and the like.

In some embodiments, provided herein is an organ mimic device inaccordance with an embodiment that contains three or more parallelchannels separated by two membranes. The organ mimic device can includeat least one mesochannel 250A and at least one microchannel 250B. Forexample, in one embodiment, one mesochannel 250A can be positionedbetween two microchannels 250B. In some embodiments, the device canfurther comprise operating channels as described herein. The overallcentral channel includes multiple membranes positioned along respectiveparallel x-y planes which separate the central channel into threedistinct central sub-channels (e.g., two microchannels and onemesochannel). The membranes can be permeable and rigid or flexible.Positive and/or negative pressurized media can be applied via operatingchannels to create a pressure differential to thereby cause themembranes to stretch and retract along their respective planes inparallel.

Details of membrane surface treatment and types of media which can beapplied to the membrane and/or through the mesochannel 250A andmicrochannel 250B in operating the device will now be discussed. Themembrane including, e.g., the porous membrane, can be coated withsubstances such as various cell adhesion promoting substances or ECMproteins, such as fibronectin, laminin, various collagen types,glycoproteins, vitronectin, elastins, fibrin, proteoglycans, heparinsulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, fibroin,chitosan, or any combinations thereof. In general, one or more celladhesion molecules is coated on one surface of the membrane 208 whereasanother cell adhesion molecule is applied to the opposing surface of themembrane 208, or both surfaces can be coated with the same cell adhesionmolecules. In some embodiments, the ECMs, which can be ECMs produced bycells, such as primary cells or embryonic stem cells, and othercompositions of matter are produced in a serum-free environment.

In an embodiment, one coats the membrane with a cell adhesion factorand/or a positively-charged molecule that are bound to the membrane toimprove cell attachment and stabilize cell growth. The positivelycharged molecule can be selected from the group consisting ofpolylysine, chitosan, poly(ethyleneimine) or acrylics polymerized fromacrylamide or methacrylamide and incorporating positively-charged groupsin the form of primary, secondary or tertiary amines, or quaternarysalts. The cell adhesion factor can be added to the membrane and isfibronectin, laminin, various collagen types, glycoproteins,vitronectin, elastins, fibrin, proteoglycans, heparin sulfate,chondroitin sulfate, keratin sulfate, hyaluronic acid, tenascin,antibodies, aptamers, or fragments or analogs having a cell bindingdomain thereof. The positively-charged molecule and/or the cell adhesionfactor can be covalently bound to the membrane. In another embodiment,the positively-charged molecule and/or the cell adhesion factor arecovalently bound to one another and either the positively-chargedmolecule or the cell adhesion factor is covalently bound to themembrane. Also, the positively-charged molecule or the cell adhesionfactor or both cam be provided in the form of a stable coatingnon-covalently bound to the membrane.

In an embodiment, the cell attachment-promoting substances,matrix-forming formulations, and other compositions of matter aresterilized to prevent unwanted contamination. Sterilization can beaccomplished, for example, by ultraviolet light, filtration, gas plasma,ozone, ethylene oxide, and/or heat. Antibiotics can also be added,particularly during incubation, to prevent the growth of bacteria, fungiand other undesired micro-organisms. Such antibiotics include, by way ofnon-limiting example, gentamicin, streptomycin, penicillin, amphotericinand ciprofloxacin.

In some embodiments, the membrane and/or other components of the devicesdescribed herein can be treated using gas plasma, charged particles,ultraviolet light, ozone, or any combinations thereof.

Cells:

In another embodiment, at least one side of the membrane is coated orcultured with cell cultures, including without limitation, primary cellcultures, established cell lines, or stem cell cultures, such as ESC,iPSCs attached to ECM substances and/or cell adhesion molecules, if any.Any prokaryotic and eukaryotic cells including, e.g., but not limitedto, human cells, animal cells, insect cells, plant cells, bacteria,fungus, and/or parasites, can be used in the devices described herein.In some embodiments, mammalian cells (e.g., a human or an animal) areused in the device described herein. Usually an animal is a vertebratesuch as a primate, rodent, domestic animal or game animal. Primatesinclude chimpanzees, cynomologous monkeys, spider monkeys, and macaques,e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbitsand hamsters. Domestic and game animals include cows, horses, pigs,deer, bison, buffalo, feline species, e.g., domestic cat, caninespecies, e.g., dog, fox, wolf, and avian species, e.g., chicken, emu,ostrich and birds. In some embodiments, the animal cells include cellsfrom fish, reptiles and amphibians. The cells can be derived from anormal healthy subject (e.g., a human or an animal) or a subject (e.g.,a human or an animal) determined to have a specific type or stage of adisease or disorder.

In accordance with some embodiments of the invention, cells can bederived from an invertebrate. For example, invertebrates can include,but are not limited to, protozoa, annelids, mollusks, crustaceans,arachnids, echinoderms, and insects.

In some embodiments, insects cells can be used in the devices describedherein. In some embodiments, plant cells can be used in the devicesdescribed herein. In some embodiments, cells derived from fungi can beused in the devices described herein. Examples of fungi can include, butare not limited to mushrooms, mold, and yeast. In accordance with someembodiments of the invention, cells derived from microorganisms can beused in the devices described herein. Examples of microorganisms caninclude, but are not limited to, bacteria and viruses.

In an embodiment, the cells attached to either side of the membrane caninclude epithelial cells, endothelial cells, fibroblasts, smooth musclecells, basal cells, ciliated cells, columnar cells, goblet cells, musclecells, immune cells, neural cells, hematopoietic cells, lung cells(e.g., alveolar epithelial cells, airway cells (e.g., small airwaycells, and large airway cells), bronchial cells, tracheal cells, andnasal epithelial cells), gut cells, brain cells, stem cells, skin cells,liver cells, heart cells, spleen cells, kidney cells, pancreatic cells,intestinal cells, keratinocytes, dermal keratinocytes, reproductivecells, blood cells (including, e.g., white blood cells, red blood cells,platelets and hematopoietic stem and progenitor cells) and anycombinations thereof. In other embodiments, the primary cells or celllines can be fibroblast cells, which include without limitation, humanfetal fibroblast cells. In some embodiments, the stem cells of the stemcell cultures are embryonic stem cells. The source of embryonic stemcells can include without limitation mammals, including non-humanprimates and humans. Non-limiting examples of human embryonic stem cellsinclude lines BG01, BG02, BG03, BG01v, CHA-hES-1, CHA-hES-2, FCNCBS1,FCNCBS2, FCNCBS3, H1, H7, H9, H13, H14, HSF-1, H9.1, H9.2, HES-1, HES-2,HES-3, HES-4, HES-5, HES-6, hES-1-2, hES-3-0, hES-4-0, hES-5-1, hES-8-1,hES-8-2, hES-9-1, hES-9-2, hES-101, hICM8, hICM9, hICM40, hICM41,hICM42, hICM43, HSF-6, HUES-1, HUES-2, HUES-3, HUES-4 HUES-5, HUES-6,HUES-7 HUES-8, HUES-9, HUES-10, HUES-11, HUES-12, HUES-13, HUES-14,HUESS-15, HUES-16, HUES-17, 13, 14, 16, 13.2, 13.3, 16.2, J3, J3.2,MB01, MB02, MB03, Miz-hES1, RCM-1, RLS ES 05, RLS ES 07, RLS ES 10, RLSES 13, RLS ES 15, RLS ES 20, RLS ES 21, SA01, SA02, and SA03. In anembodiment, the stem cells of the stem cell cultures are inducedpluripotent stem cells.

In an embodiment, the cell cultures can be cell cultures such as primarycell cultures or stem cell cultures which are serum-free. In some theseembodiments, a serum-free primary cell ECM is used in conjunction with aprimary cell serum-free medium (SFM). Suitable SFM include withoutlimitation (a) EPILIFE® Serum Free Culture Medium supplemented withEPILIFE® Defined Growth Supplement and (b) Defined Keratinocyte-SFMsupplemented with Defined Keratinocyte-SFM Growth Supplement, allcommercially available from Gibco/Invitrogen (Carlsbad, Calif., US). Insome of these embodiments, a serum-free stem cell ECM is used inconjunction with stem cell SFM. Suitable SFM include without limitationSTEMPRO® hESC Serum Free Media (SFM) supplemented with basic fibroblastgrowth factor and .beta.-mercaptoethanol, KNOCKOUT™. D-MEM supplementedwith KNOCKOUT™. Serum Replacement (SR), STEMPRO®. MSC SFM and STEMPRO®.NSC SFM, all commercially available from Gibco/Invitrogen (Carlsbad,Calif., US).

In an embodiment, the compositions can also be xeno-free. A compositionof matter is said to be “xeno-free” when it is devoid of substances fromany animal other than the species of animal from which the cells arederived. In an embodiment, the cell cultures which can be cell culturessuch as primary cell cultures or stem cell cultures are xeno-free. Inthese embodiments, a xeno-free ECM which can be an ECM such as a primarycell ECM or ECM designed specifically to support stem cell growth ordifferentiation. These matrices can be specifically designed to bexeno-free.

In an embodiment, the cell cultures are primary cells or stem cellscultured in a conditioned culture medium. In other embodiments, theculture medium is an unconditioned culture medium.

In an embodiment, the cell culture conditions are completely defined. Inthese embodiments, one uses a completely defined cell culture medium inthe fluid chambers. Suitable media include without limitation, forprimary cells, EPILIFE®. Serum Free Culture Medium supplemented withEPILIFE®. Defined Growth Supplement, and, for stem cells, STEMPRO®. hESCSFM, all commercially available from Gibco/Invitrogen, Carlsbad, Calif.,US.

To study the effects of a test agent, e.g., pharmaceuticals,environmental stressors, pathogens, toxins and such, one can add theseinto the desired cell culture medium suitable for growing the cellsattached to the membrane in the channel. Thus, one can introducepathogens, such as bacteria, viruses, aerosols, various types ofnanoparticles, toxins, gaseous substances, and such into the culturemedium which flows in the chambers to feed the cells.

A skilled artisan will also be able to control the pH balance of themedium according to the metabolic activity of the cells to maintain thepH in a suitable level for any cell or tissue type in question. Monitorsand adjustment systems to monitor and adjust pH can be inserted into thedevice.

The membrane is preferably coated on one or both sides with cells,molecules or other matter, whereby the device provides a controlledenvironment to monitor cell behavior along and/or between themesochannel and the microchannel via the membrane. One can use any cellsfrom a multicellular organism in the device. For example, human bodycomprises at least 210 known types of cells. A skilled artisan caneasily construct useful combinations of the cells in the device. Celltypes (e.g., human) which can be used in the devices include, but arenot limited to cells of the integumentary system including but notlimited to Keratinizing epithelial cells, Epidermal keratinocyte(differentiating epidermal cell), Epidermal basal cell (stem cell),Keratinocyte of fingernails and toenails, Nail bed basal cell (stemcell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticularhair shaft cell, Cuticular hair root sheath cell, Hair root sheath cellof Huxley's layer, Hair root sheath cell of Henle's layer, External hairroot sheath cell, Hair matrix cell (stem cell); Wet stratified barrierepithelial cells, such as Surface epithelial cell of stratified squamousepithelium of cornea, tongue, oral cavity, esophagus, anal canal, distalurethra and vagina, basal cell (stem cell) of epithelia of cornea,tongue, oral cavity, esophagus, anal canal, distal urethra and vagina,Urinary epithelium cell (lining urinary bladder and urinary ducts);Exocrine secretory epithelial cells, such as Salivary gland mucous cell(polysaccharide-rich secretion), Salivary gland serous cell(glycoprotein enzyme-rich secretion), Von Ebner's gland cell in tongue(washes taste buds), Mammary gland cell (milk secretion), Lacrimal glandcell (tear secretion), Ceruminous gland cell in ear (wax secretion),Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweatgland clear cell (small molecule secretion), Apocrine sweat gland cell(odoriferous secretion, sex-hormone sensitive), Gland of Moll cell ineyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebumsecretion), Bowman's gland cell in nose (washes olfactory epithelium),Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminalvesicle cell (secretes seminal fluid components, including fructose forswimming sperm), Prostate gland cell (secretes seminal fluidcomponents), Bulbourethral gland cell (mucus secretion), Bartholin'sgland cell (vaginal lubricant secretion), Gland of Littre cell (mucussecretion), Uterus endometrium cell (carbohydrate secretion), Isolatedgoblet cell of respiratory and digestive tracts (mucus secretion),Stomach lining mucous cell (mucus secretion), Gastric gland zymogeniccell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloricacid secretion), Pancreatic acinar cell (bicarbonate and digestiveenzyme secretion), pancreatic endocrine cells, Paneth cell of smallintestine (lysozyme secretion), intestinal epithelial cells, Types I andII pneumocytes of lung (surfactant secretion), and/or Clara cell oflung.

One can also coat the membrane with Hormone secreting cells, such asendocrine cells of the islet of Langerhands of the pancreas, Anteriorpituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes,Corticotropes, Intermediate pituitary cell, secretingmelanocyte-stimulating hormone; and Magnocellular neurosecretory cellssecreting oxytocin or vasopressin; Gut and respiratory tract cellssecreting serotonin, endorphin, somatostatin, gastrin, secretin,cholecystokinin, insulin, glucagon, bombesin; Thyroid gland cells suchas thyroid epithelial cell, parafollicular cell, Parathyroid glandcells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells,chromaffin cells secreting steroid hormones (mineralcorticoids and glucocorticoids), Leydig cell of testes secreting testosterone, Theca internacell of ovarian follicle secreting estrogen, Corpus luteum cell ofruptured ovarian follicle secreting progesterone, Granulosa luteincells, Theca lutein cells, Juxtaglomerular cell (renin secretion),Macula densa cell of kidney, Peripolar cell of kidney, and/or Mesangialcell of kidney.

Additionally or alternatively, one can treat at least one side of themembrane with Metabolism and storage cells such as Hepatocyte (livercell), White fat cell, Brown fat cell, Liver lipocyte. One can also useBarrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract)or Kidney cells such as Kidney glomerulus parietal cell, Kidneyglomerulus podocyte, Kidney proximal tubule brush border cell, Loop ofHenle thin segment cell, Kidney distal tubule cell, and/or Kidneycollecting duct cell.

Other cells that can be used in the device include Type I pneumocyte(lining air space of lung), Pancreatic duct cell (centroacinar cell),Nonstriated duct cell (of sweat gland, salivary gland, mammary gland,etc.), principal cell, Intercalated cell, Duct cell (of seminal vesicle,prostate gland, etc.), Intestinal brush border cell (with microvilli),Exocrine gland striated duct cell, Gall bladder epithelial cell,Ductulus efferens nonciliated cell, Epididymal principal cell, and/orEpididymal basal cell.

One can also use Epithelial cells lining closed internal body cavitiessuch as Synovial cell (lining joint cavities, hyaluronic acidsecretion), Serosal cell (lining peritoneal, pleural, and pericardialcavities), Squamous cell (lining perilymphatic space of ear), Squamouscell (lining endolymphatic space of ear), Columnar cell of endolymphaticsac with microvilli (lining endolymphatic space of ear), Columnar cellof endolymphatic sac without microvilli (lining endolymphatic space ofear), Dark cell (lining endolymphatic space of ear), Vestibular membranecell (lining endolymphatic space of ear), Stria vascularis basal cell(lining endolymphatic space of ear), Stria vascularis marginal cell(lining endolymphatic space of ear), Cell of Claudius (liningendolymphatic space of ear), Cell of Boettcher (lining endolymphaticspace of ear), Choroid plexus cell (cerebrospinal fluid secretion),Pia-arachnoid squamous cell, Pigmented ciliary epithelium cell of eye,Nonpigmented ciliary epithelium cell of eye.

The following cells can be used in the device by adding them to thesurface of the membrane in culture medium. These cells include cellssuch as Ciliated cells with propulsive function such as Respiratorytract ciliated cell, Oviduct ciliated cell (in female), Uterineendometrial ciliated cell (in female), Rete testis ciliated cell (inmale), Ductulus efferens ciliated cell (in male), and/or Ciliatedependymal cell of central nervous system (lining brain cavities).

One can also plate cells that secrete specialized ECMs, such asAmeloblast epithelial cell (tooth enamel secretion), Planum semilunatumepithelial cell of vestibular apparatus of ear (proteoglycan secretion),Organ of Corti interdental epithelial cell (secreting tectorial membranecovering hair cells), Loose connective tissue fibroblasts, Cornealfibroblasts (corneal keratocytes), Tendon fibroblasts, Bone marrowreticular tissue fibroblasts, Other nonepithelial fibroblasts, Pericyte,Nucleus pulposus cell of intervertebral disc, Cementoblast/cementocyte(tooth root bonelike cementum secretion), Odontoblast/odontocyte (toothdentin secretion), Hyaline cartilage chondrocyte, Fibrocartilagechondrocyte, Elastic cartilage chondrocyte, Osteoblast/osteocyte,Osteoprogenitor cell (stem cell of osteoblasts), Hyalocyte of vitreousbody of eye, Stellate cell of perilymphatic space of ear, Hepaticstellate cell (Ito cell), and/or Pancreatic stellate cell.

Additionally or alternatively, contractile cells, such as Skeletalmuscle cells, Red skeletal muscle cell (slow), White skeletal musclecell (fast), Intermediate skeletal muscle cell, nuclear bag cell ofmuscle spindle, nuclear chain cell of muscle spindle, Satellite cell(stem cell), Heart muscle cells, Ordinary heart muscle cell, Nodal heartmuscle cell, Purkinje fiber cell, Smooth muscle cell (various types),Myoepithelial cell of iris, Myoepithelial cell of exocrine glands can beused in the present device.

The following cells can also be used in the present device: Blood andimmune system cells, such as Erythrocyte (red blood cell), Megakaryocyte(platelet precursor), Monocyte, Connective tissue macrophage (varioustypes), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell(in lymphoid tissues), Microglial cell (in central nervous system),Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte,Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, NaturalKiller T cell, B cell, Natural killer cell, Reticulocyte, Stem cells andcommitted progenitors for the blood and immune system (various types).One can use these cells as single cell types or in mixtures to determineeffects of the immune cells in the tissue culture system.

One can also treat the membranes with one or more Nervous system cells,Sensory transducer cells such as Auditory inner hair cell of organ ofCorti, Auditory outer hair cell of organ of Corti, Basal cell ofolfactory epithelium (stem cell for olfactory neurons), Cold-sensitiveprimary sensory neurons, Heat-sensitive primary sensory neurons, Merkelcell of epidermis (touch sensor), Olfactory receptor neuron,Pain-sensitive primary sensory neurons (various types); Photoreceptorcells of retina in eye including Photoreceptor rod cells, Photoreceptorblue-sensitive cone cell of eye, Photoreceptor green-sensitive cone cellof eye, Photoreceptor red-sensitive cone cell of eye, Proprioceptiveprimary sensory neurons (various types); Touch-sensitive primary sensoryneurons (various types); Type I carotid body cell (blood pH sensor);Type II carotid body cell (blood pH sensor); Type I hair cell ofvestibular apparatus of ear (acceleration and gravity); Type II haircell of vestibular apparatus of ear (acceleration and gravity); and/orType I taste bud cell.

One can further use Autonomic neuron cells such as Cholinergic neuralcell (various types), Adrenergic neural cell (various types),Peptidergic neural cell (various types) in the present device. Further,sense organ and peripheral neuron supporting cells can also be used.These include, for example, Inner pillar cell of organ of Corti, Outerpillar cell of organ of Corti, Inner phalangeal cell of organ of Corti,Outer phalangeal cell of organ of Corti, Border cell of organ of Corti,Hensen cell of organ of Corti, Vestibular apparatus supporting cell.Type I taste bud supporting cell, Olfactory epithelium supporting cell,Schwann cell, Satellite cell (encapsulating peripheral nerve cellbodies) and/or Enteric glial cell. In some embodiments, one can also usecentral nervous system neurons and glial cells such as Astrocyte(various types), Neuron cells (large variety of types, still poorlyclassified), Oligodendrocyte, and Spindle neuron.

Lens cells such as Anterior lens epithelial cell andCrystallin-containing lens fiber cell can also be used in the presentdevice. Additionally, one can use pigment cells such as melanocytes andretinal pigmented epithelial cells; and germ cells, such asOogonium/Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cellfor spermatocyte), and Spermatozoon.

In some embodiments one can add to the membrane nurse cells Ovarianfollicle cell, Sertoli cell (in testis), Thymus epithelial cell. One canalso use interstitial cells such as interstitial kidney cells.

In an embodiment, one can coat at least one side of the membrane withepithelial cells. Epithelium is a tissue composed of cells that line thecavities and surfaces of structures throughout the body. Many glands arealso formed from epithelial tissue. It lies on top of connective tissue,and the two layers are separated by a basement membrane. In humans,epithelium is classified as a primary body tissue, the other ones beingconnective tissue, muscle tissue and nervous tissue. Epithelium is oftendefined by the expression of the adhesion molecule e-cadherin (asopposed to n-cadherin, which is used by neurons and cells of theconnective tissue).

Functions of epithelial cells include secretion, selective absorption,protection, transcellular transport and detection of sensation and theycommonly as a result present extensive apical-basolateral polarity (e.g.different membrane proteins expressed) and specialization. Examples ofepithelial cells include squamous cells that have the appearance ofthin, flat plates. They fit closely together in tissues; providing asmooth, low-friction surface over which fluids can move easily. Theshape of the nucleus usually corresponds to the cell form and helps toidentify the type of epithelium. Squamous cells tend to havehorizontally flattened, elliptical nuclei because of the thin flattenedform of the cell. Classically, squamous epithelia are found liningsurfaces utilizing simple passive diffusion such as the alveolarepithelium in the lungs. Specialized squamous epithelia also form thelining of cavities such as the blood vessels (endothelium) and heart(mesothelium) and the major cavities found within the body.

Another example of epithelial cells is cuboidal cells. Cuboidal cellsare roughly cuboidal in shape, appearing square in cross section. Eachcell has a spherical nucleus in the centre. Cuboidal epithelium iscommonly found in secretive or absorptive tissue: for example the(secretive) exocrine gland the pancreas and the (absorptive) lining ofthe kidney tubules as well as in the ducts of the glands. They alsoconstitute the germinal epithelium which produces the egg cells in thefemale ovary and the sperm cells in the male testes.

Yet another type of epithelial cells are columnar epithelial cells thatare elongated and column-shaped. Their nuclei are elongated and areusually located near the base of the cells. Columnar epithelium formsthe lining of the stomach and intestines. Some columnar cells arespecialised for sensory reception such as in the nose, ears and thetaste buds of the tongue. Goblet cells (unicellular glands) are foundbetween the columnar epithelial cells of the duodenum. They secretemucus, which acts as a lubricant.

Still another example of the epithelial cells are pseudostratifiedcells. These are simple columnar epithelial cells whose nuclei appear atdifferent heights, giving the misleading (hence “pseudo”) impressionthat the epithelium is stratified when the cells are viewed in crosssection. Pseudostratified epithelium can also possess fine hair-likeextensions of their apical (luminal) membrane called cilia. In thiscase, the epithelium is described as “ciliated” pseudostratifiedepithelium. Cilia are capable of energy dependent pulsatile beating in acertain direction through interaction of cytoskeletal microtubules andconnecting structural proteins and enzymes. The wafting effect producedcauses mucus secreted locally by the goblet cells (to lubricate and totrap pathogens and particles) to flow in that direction (typically outof the body). Ciliated epithelium is found in the airways (nose,bronchi), but is also found in the uterus and Fallopian tubes offemales, where the cilia propel the ovum to the uterus.

Epithelium lines both the outside (skin) and the inside cavities andlumen of bodies. The outermost layer of our skin is composed of deadstratified squamous, keratinised epithelial cells.

Tissues that line the inside of the mouth, the oesophagus and part ofthe rectum are composed of nonkeratinized stratified squamousepithelium. Other surfaces that separate body cavities from the outsideenvironment are lined by simple squamous, columnar, or pseudostratifiedepithelial cells. Other epithelial cells line the insides of the lungs,the gastrointestinal tract, the reproductive and urinary tracts, andmake up the exocrine and endocrine glands. The outer surface of thecornea is covered with fast-growing, easily-regenerated epithelialcells. Endothelium (the inner lining of blood vessels, the heart, andlymphatic vessels) is a specialized form of epithelium. Another type,mesothelium, forms the walls of the pericardium, pleurae, andperitoneum.

Accordingly, one can recreate any of these tissues in the cell culturedevice as described by plating applicable cell types on the membranesand/or applying applicable mechanical modulation of the membrane toprovide physiological or artificial mechanical force on the cells tomimic physiological forces, such as tension on skin or mechanical strainon lung. In an embodiment, one side of the membrane is coated withepithelial cells and the other side is coated with endothelial cells.Examples of endothelial cells include, but are not limited to, bloodvessel and lymphatic vascular endothelial fenestrated cell, blood vesseland lymphatic vascular endothelial continuous cell, blood vessel andlymphatic vascular endothelial splenic cell, corneal endothelial cell,and any combinations thereof.

The endothelium is the thin layer of cells that line the interiorsurface of blood vessels and lymphatic vessels, forming an interfacebetween circulating blood or lymph in the lumen and the rest of thevessel wall. Endothelial cells in direct contact with blood are vascularendothelial cells, whereas those in direct contact with lymph are knownas lymphatic endothelial cells. Endothelial cells line the entirecirculatory system, from the heart to the smallest capillary. Thesecells reduce turbulence of the flow of blood allowing the fluid to bepumped farther.

The foundational model of anatomy makes a distinction betweenendothelial cells and epithelial cells on the basis of which tissuesthey develop from and states that the presence of vimentin rather thankeratin filaments separate these from epithelial cells. Endothelium ofthe interior surfaces of the heart chambers are called endocardium. Bothblood and lymphatic capillaries are composed of a single layer ofendothelial cells called a monolayer. Endothelial cells are involved inmany aspects of vascular biology, including: vasoconstriction andvasodilation, and hence the control of blood pressure; blood clotting(thrombosis & fibrinolysis); atherosclerosis; formation of new bloodvessels (angiogenesis); inflammation and barrier function—theendothelium acts as a selective barrier between the vessel lumen andsurrounding tissue, controlling the passage of materials and the transitof white blood cells into and out of the bloodstream. Excessive orprolonged increases in permeability of the endothelial monolayer, as incases of chronic inflammation, can lead to tissue edema/swelling. Insome organs, there are highly differentiated endothelial cells toperform specialized ‘filtering’ functions. Examples of such uniqueendothelial structures include the renal glomerulus and the blood-brainbarrier.

In an embodiment, the membrane side that contains cultured endothelialcells can be exposed to various test substances and also white bloodcells or specific immune system cells flowing in the bottom microchannelto study effects of the test agents on the function of the immune systemcells at the tissue level.

The devices described herein can be provided with pre-seeded cells or apre-formed tissue structure, or without pre-seeded cells.

Using the organ mimic device described herein, one can studybiotransformation, absorption, clearance, metabolism, and activation ofxenobiotics, as well as drug delivery. The bioavailability and transportof chemical and biological agents across epithelial layers as in theintestine, endothelial layers as in blood vessels, and across theblood-brain barrier can also be studied. The acute basal toxicity, acutelocal toxicity or acute organ-specific toxicity, teratogenicity,genotoxicity, carcinogenicity, and mutagenicity, of chemical agents canalso be studied. Effects of infectious biological agents, biologicalweapons, harmful chemical agents and chemical weapons can also bedetected and studied. Infectious diseases and the efficacy of chemicaland biological agents to treat these diseases, as well as optimal dosageranges for these agents, can be studied. The response of organs in vivoto chemical and biological agents, and the pharmacokinetics andpharmacodynamics of these agents can be detected and studied using thepresent device. The impact of genetic content on response to the agentscan be studied. The amount of protein and gene expression in response tochemical or biological agents can be determined. Changes in metabolismin response to chemical or biological agents can be studied as wellusing the present device.

The advantages of the organ mimic device, as opposed to conventionalcell cultures or tissue cultures are numerous. For instance, when cellsare placed in the organ mimic device, fibroblast, SMC (smooth musclecell), endothelial cells, and/or epithelial cell differentiation canoccur that reestablishes a defined three-dimensional architecturaltissue-tissue relationships that are close to the in vivo situation, andcell functions and responses to pharmacological agents or activesubstances or products can be investigated at the tissue and organlevels.

In addition, many cellular or tissue activities are amenable todetection in the organ mimic device, including, but not limited to,diffusion rate of the drugs into and through the layered tissues intransported flow channel; cell morphology, differentiation and secretionchanges at different layers; cell locomotion, growth, apoptosis, and thelike. Further, the effect of various drugs on different types of cellslocated at different layers of the system can be assessed easily.

For drug discovery, for example, there can be two advantages for usingthe organ mimic device described herein: (1) the organ mimic device isbetter able to mimic in vivo layered architecture of tissues andtherefore allow one to study drug effect at the organ level in additionto at the cellular and tissue levels; and (2) the organ mimic devicedecreases the use of in vivo tissue models and the use of animals fordrug selection and toxicology studies.

In addition to drug discovery and development, the organ mimic devicedescribed herein can be also useful in basic and clinical research. Forexample, the organ mimic device can be used to research the mechanism oftumorigenesis. It is well established that in vivo cancer progression ismodulated by the host and the tumor micro-environment, including thestromal cells and extracellular matrix (ECM). For example, stromal cellswere found being able to convert benign epithelial cells to malignantcells, thereby ECM was found to affect the tumor formation. There isgrowing evidence that cells growing in defined architecture are moreresistant to cytotoxic agents than cells in mono layers. Therefore, anorgan mimic device is a better means for simulating the original growthcharacteristics of cancer cells and thereby better reflects the realdrug's sensitivity of in vivo tumors.

The organ mimic device can be employed in engineering a variety oftissues including, but not limited to, the cardiovascular system, lung,intestine, kidney, brain, bone marrow, bones, teeth, and skin. If thedevice is fabricated with a suitable biocompatible and/or biodegradablematerial, such as poly-lactide-co-glycolide acid (PLGA), the organ mimicdevice can be used for transplantation or implantation in vivo.Moreover, the ability to spatially localize and control interactions ofseveral cell types presents an opportunity to engineer hierarchically,and to create more physiologically correct tissue and organ analogs. Thearrangement of multiple cell types in defined arrangement has beneficialeffects on cell differentiation, maintenance, and functional longevity.

The organ mimic device can also allow different growth factors,chemicals, gases and nutrients to be added to different cell typesaccording to the needs of cells and their existence in vivo. Controllingthe location of those factors or proteins can direct the process ofspecific cell remodeling and functioning, and also can provide themolecular cues to the whole system, resulting in such beneficialdevelopments as neotissue, cell remodeling, enhanced secretion, and thelike.

In yet another aspect, the organ mimic device can be utilized as multicell type cellular microarrays, such as microfluidic devices. Using theorgan mimic device, pattern integrity of cellular arrays can bemaintained. These cellular microarrays can constitute the future“lab-on-a-chip”, particularly when multiplexed and automated. Theseminiaturized multi cell type cultures will facilitate the observation ofcell dynamics with faster, less noisy assays, having built-in complexitythat will allow cells to exhibit in vivo-like responses to the array.

In yet another aspect, the organ mimic device can be utilized asbiological sensors. Cell-based biosensors can provide more informationthan other biosensors because cells often have multifacetedphysiological responses to stimuli, as well as novel mechanisms toamplify these responses. All cell types in the organ mimic device can beused to monitor different aspects of an analyte at the same time;different cell type in the organ mimic device can be used to monitordifferent analytes at the same time; or a mixture of both types ofmonitoring. Cells ranging from E. coli to cells of mammalian lines havebeen used as sensors for applications in environmental monitoring, toxindetection, and physiological monitoring.

In yet another aspect, the organ mimic device can be used inunderstanding fundamental processes in cell biology and cell-ECMinteractions. The in vivo remodeling process is a complicated, dynamic,reciprocal process between cells and ECMs. The organ mimic device wouldbe able to capture the complexity of these biological systems, renderingthese systems amenable to investigation and beneficial manipulation.Furthermore, coupled with imaging tools, such as fluorescencemicroscopy, microfluorimetry or optical coherence tomography (OCT),real-time analysis of cellular behavior in the multilayered tissues isexpected using the device. Examples of cell and tissue studies amenableto real-time analysis include cell secretion and signaling, cell-cellinteractions, tissue-tissue interactions, dynamic engineered tissueconstruction and monitoring, structure-function investigations in tissueengineering, and the process of cell remodeling matrices in vitro.

Another example of the use of this device is to induce tissue-tissueinterfaces and complex organ structures to form within the device byimplanting it in vivo within the body of a living animal, and allowingcells and tissues to impregnate the device and establish normaltissue-tissue interfaces. Then the whole device and contained cells andtissues is surgically removed while perfusing it through one or more ofthe fluid channels with medium and gases necessary for cell survival.This complex organ mimic can then be maintained viable in vitro throughcontinuous perfusion and used to study highly complex cell and tissuefunctions in their normal 3D context with a level of complexity notpossible using any existing in vitro model system.

In particular, a microchannel device can be implanted subcutaneously invivo into an animal in which the device contains bone-inducingmaterials, such as demineralized bone powder or bone morphogenicproteins (BMPs), in a channel that has one or more corresponding portsopen to the surrounding tissue space. The second channel would be closedduring implantation by closing its end ports or filling it with a solidremovable material, such as a solid rod. As a result of wound healing,connective tissues containing microcapillaries and mesenchymal stemcells would grow into the open channels of the device and, due to thepresence of the bone-inducing material, will form bone with spaces thatrecruit circulating hematopoietic precursor cells to form fullyfunctional bone marrow, as shown in past studies.

Once this process is complete, the surgical site would be reopened, andthe second channel would be reopened by removing the rod or plugs andwould then be connected with catheters linked to a fluid reservoir sothat culture medium containing nutrients and gases necessary for cellsurvival could be pumped through the second channel and passed throughthe pores of the membrane into the first channel containing the formedbone marrow. The entire microchannel device could then be cut free fromthe surrounding tissue, and with the medium flowing continuously intothe device, would be removed from the animal and passed to a tissueculture incubator and maintained in culture with continuous fluid flowthrough the second channel, and additional flow can be added to thefirst channel as well if desired by connecting to their inlet and outletports. The microchannel device would then be used to study intact bonemarrow function in vitro as in a controlled environment.

Both biocompatible and biodegradable materials can be used in thepresent device to facilitate in vivo implantation of the present device.One can also use biocompatible and biodegradable coatings. For example,one can use ceramic coatings on a metallic substrate. But any type ofcoating material and the coating can be made of different types ofmaterials: metals, ceramics, polymers, hydrogels or a combination of anyof these materials.

Biocompatible materials include, but are not limited to an oxide, aphosphate, a carbonate, a nitride or a carbonitride. Among the oxide thefollowing ones are preferred: tantalum oxide, aluminum oxide, iridiumoxide, zirconium oxide or titanium oxide. In some cases the coating canalso be made of a biodegradable material that will dissolve over timeand can be replaced by the living tissue. Substrates are made ofmaterials such as metals, ceramics, polymers or a combination of any ofthese. Metals such as stainless steel, Nitinol, titanium, titaniumalloys, or aluminum and ceramics such as zirconia, alumina, or calciumphosphate are of particular interest.

The biocompatible material can also be biodegradable in that it willdissolve over time and can be replaced by the living tissue. Suchbiodegradable materials include, but are not limited to, poly(lacticacid-co-glycolic acid), polylactic acid, polyglycolic acid (PGA),collagen or other ECM molecules, other connective tissue proteins,magnesium alloys, polycaprolactone, hyaluric acid, adhesive proteins,biodegradable polymers, synthetic, biocompatible and biodegradablematerial, such as biopolymers, bioglasses, bioceramics, calcium sulfate,calcium phosphate such as, for example, monocalcium phosphatemonohydrate, monocalcium phosphate anhydrous, dicalcium phosphatedihydrate, dicalcium phosphate anhydrous, tetracalcium phosphate,calcium orthophosphate phosphate, calcium pyrophosphate,alpha-tricalcium phosphate, beta-tricalcium phosphate, apatite such ashydroxyapatite, or polymers such as, for example,poly(alpha-hydroxyesters), poly(ortho esters), poly(ether esters),polyanhydrides, poly(phosphazenes), poly(propylene fumarates),poly(ester amides), poly(ethylene fumarates), poly(amino acids),polysaccharides, polypeptides, poly(hydroxy butyrates), poly(hydroxyvalerates), polyurethanes, poly(malic acid), polylactides,polyglycolides, polycaprolactones, poly(glycolide-co-trimethylenecarbonates), polydioxanones, or copolymers, terpolymers thereof orblends of those polymers, or a combination of biocompatible andbiodegradable materials. One can also use biodegradable glass andbioactive glass self-reinforced and ultrahigh strength bioabsorbablecomposites assembled from partially crystalline bioabsorbable polymers,like polyglycolides, polylactides and/or glycolide/lactide copolymers.

These materials preferably have high initial strength, appropriatemodulus and strength retention time from 4 weeks up to 1 year in vivo,depending on the implant geometry. Reinforcing elements such as fibersof crystalline polymers, fibers of carbon in polymeric resins, andparticulate fillers, e.g., hydroxyapatite, can also be used to providethe dimensional stability and mechanical properties of biodegradabledevices. The use of interpenetrating networks (IPN) in biodegradablematerial construction has been demonstrated as a means to improvemechanical strength. To further improve the mechanical properties ofIPN-reinforced biodegradable materials, the present device can beprepared as semi-interpenetrating networks (SIPN) of crosslinkedpolypropylene fumarate within a host matrix ofpoly(lactide-co-glycolide) 85:15 (PLGA) orpoly(l-lactide-co-d,l-lactide) 70:30 (PLA) using different crosslinkingagents. One can also use natural poly(hydroxybutyrate-co-9%hydroxyvalerate) copolyester membranes as described in Charles-HilaireRivard et al. (Journal of Applied Biomaterials, Volume 6 Issue 1, Pages65-68, 1 Sep. 2004). A skilled artisan will be able to also select otherbiodegradable materials suitable for any specific purposes and cell andtissue types according to the applications in which the device is used.

The device as described can also be used as therapeutic devices, whenplaced in vivo. One can re-create organ mimics, such as bone marrow orlymph nodes by placing the devices in, for example an animal modelallowing the device to be inhabited by living cells and tissues, andthen removing the entire device with living cells while perfusing thevascular channel with medium. The device can then be removed and keptalive ex vivo for in vitro or ex vivo studies. In particular, themembrane can be coated with one or more cell layers on at least one sideof the membrane in vitro. In this embodiment, the cells are platedoutside an organism. In an embodiment, the membrane is coated with oneor more cell layers on at least one side of the membrane in vivo. Onecan treat one side of the membrane in vitro and the other side in vivo.One can also have one or both sides initially coated with one cell typein vitro and then implant the device to attract additional cell layersin vivo.

Additional Examples of Tissue/Organ-Mimic Devices

In some embodiments, the devices described herein can be adapted tomodel at least a portion of a tissue or organ that requires a tallerchannel to accommodate formation of a stratified, pesudostratified orthree-dimensional structure, and/or provide sufficient overhead space topermit low shear stress produced by air and/or liquid flow over thecells in order to simulate a native physiological environment. Withoutwishing to be limiting, one of skill in the art will readily appreciatethat the devices described herein can also be used to model at least aportion of a tissue or organ that does not necessarily require suchadditional space for optimum cell growth and/or fluid flow. In theseembodiments, the air/fluid flow can be adjusted to account for anincreased overhead space over the cells in order to maintain aphysiologically-relevant shear level subjected to the cells.

In some embodiments, the devices described herein can be used to modelat least a portion of a skin tissue or organ, which can be in turn usedto study or mimic a skin-related physiologically-relevant condition(e.g., a normal and/or pathological condition) for various applicationsdescribed herein. The taller mesochannel can be used to provide morespace for multiple layers of cells and/or structures as they mature ordifferentiate. Examples of a skin-related disease or disorder that canbe modeled using the devices described herein include, but are notlimited to, aging, atopic dermatitis, contact dermatitis (allergy orirritant), eczema, psoriasis, acne, epidermal hyperkeratosis,acanthosis, epidermal inflammation, dermal inflammation or pruritus,rosacea, netherton syndrome, peeling skin syndrome type A and B,hereditary ichtyosis, hidradenitis suppurativa, erythroderma(generalized exfoliative dermatitis), skin cancer, and any combinationsthereof. This can also be used to study absorption, efficacy and/ortoxicity of topically applied cosmetics or consumer products. In someembodiments, the devices described herein can be used to model an agingskin. This can also be used to study transdermal drug delivery.

A mammalian skin is generally composed of two primary layers: theepidermis, which provides a protective barrier; and the dermis, which isthe layer of skin beneath the epidermis. The epidermis is a stratifiedsquamous epithelium comprising multiple cell layers, namely (beginningwith the outermost layer), stratum corneum, stratum lucidum (primarilyin palms and soles), stratum granulosum, stratum spinosum, stratumgerminativum (also known as stratum basale). Keratinocytes constitute amajority of the epidermis, while Merkel cells, melanocytes, andLangerhans cells are also present.

The dermis layer is primarily composed of connective tissue andextracelluar matrix (e.g., collagen fibrils, microfibrils, and elasticfibers) which provide tensile strength and elasticity to the skin. Thedermis layer also harbors many mechanoreceptors (e.g., nerve endings)that provide sense of touch and heat. It also contains hair follicles,sweat glands, sebaceous glands, apocrine glands, lymphatic vessels andblood vessels. The blood vessels in the dermis can provide nourishmentand/or waste removal from its own cells as well as for the epidermis.

The dermis is connected to the epidermis through a basement membrane andis structurally divided into two areas: a superficial area adjacent tothe epidermis, called the papillary region, and a deep thicker areaknown as the reticular region. The papillary region contains looseareolar connective tissue and fingerlike projections (known as papillae)that extend toward the epidermis. The papillae provide the dermis with a“bumpy” surface that interdigitates with the epidermis, strengtheningthe connection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. The reticularregion contains dense irregular connective tissue, and a denseconcentration of collagenous, elastic, and reticular fibers that weavethroughout it. These protein fibers give the dermis its properties ofstrength, extensibility, and elasticity. The other component of thedermis that can be critical for skin physiology and/or pathophysiologyincludes the vasculature.

Accordingly, the mesochannel 250A can have a height dimension configuredto permit formation of a skin equivalent that mimics the skin of a humanor an animal. In some embodiments, the membrane of the device can beused as a basement separating the epidermis layer and dermis layer. Forexample, the surface of the membrane facing the mesochannel can becoated with keratinocytes (and optionally other cells that are typicallypresent in an epidermis such as Merkel cells, melanocytes, andLangerhans cells). The keratinocytes on the membrane can be cultured atan air-liquid interface (in a similar setup as shown in the “smallairway” example) to induce cell differentiation for formation of thestratified epidermis layer. During the differentiation process, thekeratinocytes can become highly organized, form cellular junctions (tomimic desmosomes) between each other and/or secrete keratin proteinsand/or lipids which can contribute to the formation of an extracellularmatrix and provide mechanical strength. In some embodiments,keratinocytes from the outermost stratified layer can eventually shedfrom the epidermis, as keratinocytes shed from the stratum corneum invivo.

While in some embodiments, the epidermis layer and dermis layer can beformed in the mesochannel and microchannel, respectively, in someembodiments, both the epidermis layer and dermis layer can be formed inthe mesochannel.

The other surface of the membrane facing the microchannel can be coatedwith or without cells. In some embodiments, the surface of the membranefacing the microchannel can be coated with cells selected from the groupconsisting of cell types that are typically present in a dermis layer(e.g., fibroblasts), hypodermis-associated cells (e.g., fibroblasts,macrophages, and/or adipocytes), blood vessel-associated cells asdescribed herein, and any combinations thereof.

In some embodiments where the surface of the membrane facing themicrochannel is used to model a dermis layer, the other surface of themembrane facing the mesochannel can be coated with or withoutepidermis-associated cells. In these embodiments, the membrane of thedevice can be used as a protective barrier as the epidermis layer.

In some embodiments, microorganisms typically present on a skin surface,e.g., Staphylococcus epidermis, can be cultured with the epidermis layerformed in the mesochannel.

In some embodiments, the membrane used in the device for modeling a skintissue can be porous and flexible. In some embodiments, the membrane canbe mechanically modulated by a pneumatic mechanism and/or mechanicalmeans as described herein, for example, to mimic a mechanical staticstrain typically experienced by skin cells in vivo.

In some embodiments, the devices described herein can be used to modelat least a portion of a heart. In accordance with some embodiments ofthe invention, the heart-mimic device can be used to study or mimic aheart-related physiologically-relevant condition (e.g., a normal and/orpathological condition) for various applications described herein.Examples of a heart-related disease or disorder that can be modeledusing the devices described herein include, but are not limited to,coronary heart disease (also ischemic heart disease or coronary arterydisease), cardiomyopathy, hypertensive heart disease, heart failure, corpulmonale, cardiac dysrhythmias, inflammatory heart disease (e.g.,endocarditis, inflammatory cardiomegaly, and myocarditis), valvularheart disease, congenital heart disease, rheumatic heart disease,atherosclerosis, and any combinations thereof. It can also be used tostudy effects of drugs or toxins on normal heart viability and/orfunction.

For example, in some embodiments, the surface of the membrane facing themesochannel can be coated with thin films of functional heart tissues,while the other surface facing the microchannel can be coated with orwithout blood vessel-associated cells as described herein. In someembodiments, thin films of functional heart tissues can be fabricatedfirst and then placed on the membrane facing the mesochannel. Forexample, thin films of functional heart tissues can be fabricated byculturing cardiomyocytes (e.g., ventricular cardiomyocytes) onelastomeric polymer thin films micropatterned with cell adhesionproteins (e.g., extracellular matrix proteins) to promote spatiallyordered, two-dimensional myogenesis and thus create “muscular thinfilms” (MTFs) as previously described, e.g., in Grosberg A. et al.“Ensembles of engineered cardiac tissues for physiological andpharmacological study: Heart on a chip” Lab on a chip (2011) 11: 4165,as well as the International Application Nos. WO 2008/051265, andWO2010/042856, the contents of which are incorporated herein byreference. These muscular thin films can be electrically functional andactively contractile, generating stresses comparable to those producedby whole papillary muscle. For example, the cardiomyocytes on themuscular thin films can contract, causing the elastomeric polymer thinfilms to bend and form a three-dimensional (3D) structure. Accordingly,in some embodiments, the mesochannel 250A can have a height dimensionsufficient to accommodate the height of the muscular thin films as theybend and form a 3D structure.

In some embodiments, contractile heart muscle cells (e.g.,cardiomyocytes) can be grown on a surface of a flexible and porousmembrane facing the mesochannel, while the other surface facing themicrochannel can be coated with or without blood vessel-associated cellsas described herein. As the heart muscle cells contract, the poreapertures on the membrane can deform due to cell contraction. By way ofexample only, the pore apertures can remain as a circle when the heartmuscle cells are in a relaxed state, but the circular pore aperturesbecome deformed, e.g., becoming an oval, or an ellipse, due to musclecell contraction. See, e.g., International Patent Application:PCT/US12/68766 filed Dec. 10, 2012, the content of which is incorporatedherein by reference. In this embodiment, a taller mesochannel canprovide low shear stress to heart muscle cells as in a nativephysiological microenvironment.

In some embodiments, myoblasts can be grown on the membrane facing themesochannel (with or without mechanical modulation of the membrane) toinduce differentiation of the myoblasts to form myocytes orcardiomyocytes.

In some embodiments, the devices described herein can be used to modelat least a portion of an eye, which can be in turn used to study ormimic an ocular condition (e.g., a normal and/or pathological condition)for various applications described herein. The taller mesochannel canprovide low shear stress to the delicate ocular cells as in a nativephysiological microenvironment. Examples of an ocular disease ordisorder associated with the front and/or back of an eye that can bemodeled using the devices described herein include, but are not limitedto, age-related macular degeneration, choroidal neovascularization,diabetic macular edema, acute and chronic macular neuroretinopathy,central serous chorioretinopathy, macular edema, acute multifocalplacoid pigment epitheliopathy, Behcet's disease, birdshotretinochoroidopathy, posterior uveitis, posterior scleritis, serpignouschoroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Haradasyndrome, retinal arterial occlusive disease, central retinal veinocclusion, disseminated intravascular coagulopathy, branch retinal veinocclusion, hypertensive fundus changes, ocular ischemic syndrome,retinal arterial microaneurysms, Coat's disease, parafovealtelangiectasis, hemi-retinal vein occlusion, papillophlebitis, carotidartery disease (CAD), frosted branch angitis, sickle cell retinopathy,angioid streaks, familial exudative vitreoretinopathy, Eales disease,proliferative vitreal retinopathy, diabetic retinopathy, retinal diseaseassociated with tumors, congenital hypertrophy of the retinal pigmentepithelium (RPE), posterior uveal melanoma, choroidal hemangioma,choroidal osteoma, choroidal metastasis, combined hamartoma of theretina and retinal pigmented epithelium, retinoblastoma,vasoproliferative tumors of the ocular fundus, retinal astrocytoma,intraocular lymphoid tumors, myopic retinal degeneration, acute retinalpigment epithelitis, glaucoma, endophthalmitis, cytomegalovirusretinitis, retinal cancers, and any combinations thereof. This can alsobe used to study ocular drug delivery.

In some embodiments, the devices described herein can be used to modelat least a front portion of an eye. For example, in some embodiments,the surface of the membrane facing the mesochannel can be coated withcornea-associated cells (e.g., but not limited to, corneal epithelialcells, corneal keratocytes, and/or corneal nerve cells), while the othersurface of the membrane can be optionally lined by corneal endothelialcells, with or without corneal fibroblasts included as an interveninglayer. A liquid fluid with a similar viscosity as aqueous humor can flowthrough the microchannel to provide nutrients to the cells in themesochannel, while the cells are cultured at an air-liquid interface (ina similar setup as in the “small airway” example). This model can beused to study immune response of the cornea.

In some embodiments, the devices described herein can be used to modelat least a back portion of an eye, e.g., a portion of a retina. Retinais a light-sensitive layered structure with several layers of neurons, aphotoreceptor layer (e.g., comprising rod and/or cone cells) and aretinal pigment epithelium (e.g., comprising cuboidal cells).Accordingly, in some embodiments, the surface of the membrane facing themesochannel can be coated with at least one or more layers (including,e.g., at least two or more layers) of retina-associated cells. Forexample, in one embodiment, the surface of the membrane facing themesochannel can be coated with a bottom layer of retinal epithelialcells overlaid with at least one cell layer comprising photoreceptorcells (e.g., rod and/or cone cells). The other surface of the membranefacing the microchannel can be coated with or without bloodvessel-associated cells as described herein.

In some embodiments, a liquid fluid with a viscosity as vitreous humorcan flow through the mesochannel, while a liquid fluid, e.g., cellculture medium and/or blood, can flow through the microchannel.

The retina tissue-mimic device can be used to model a retina-associateddisease, including, e.g., but not limited to, retinitis pigmentosa,macular degeneration, cone-rod dystrophy (CORD), hypertensiveretinopathy, diabetic retinopathy, retinoblastoma, retinal dysplasia,progressive retinal atrophy, and any combinations thereof.

In some embodiments, the membrane used in the device to model a front orback portion of a tissue can be porous and rigid or flexible.

In addition to modeling a portion of an intestine (e.g., a small orlarge intestine) as described earlier, in some embodiments, the devicesdescribed herein can be used to model at least a portion of an organassociated with a gastrointestinal tract or a digestive system,including, e.g., but not limited to, oropharynx, stomach, esophagus,pancreas, rectum and anus. In some embodiments, the devices describedherein can be used to model at least a portion of a pancreatic tissue,which can be in turn used to study or mimic a pancreas-relatedphysiologically-relevant condition (e.g., a normal and/or pathologicalcondition) for various applications described herein. The tallermesochannel can provide low shear stress to pancreas-associated cells,such as endocrine islet beta cells or exocrine acinar cells, as in anative physiological environment, optionally along with vascularendothelial cells lining the opposite side of the porous membrane undernormal hemodynamic flow conditions. Examples of a pancreas-relateddisease or disorder that can be modeled using the devices describedherein include, but are not limited to, diabetes, pancreatitis, cysticfibrosis, pancreatic cancer, and any combinations thereof.

In some embodiments, the surface of the membrane facing the mesochannelcan be coated with pancreas-associated cells (e.g., islets of Langerhansor endocrine cells and/or acinar cells), while the other surface of themembrane facing the microchannel can be coated with or without bloodvessel-associated cells.

In some embodiments, the membrane used in the device to model apancreatic tissue can be porous and rigid or flexible.

Use of the devices described herein to model various specific tissuesare provided herein as illustrative examples and are not intended to bein any way limiting. Those of skill in the art will realize that thedevices described herein can be adapted to mimic function of any portionof a tissue or organ in any living organisms, e.g., vertebrates (e.g.,but not limited to, human subjects or animals such as fish, birds,reptiles, and amphibians), invertebrates (e.g., but not limited to,protozoa, annelids, mollusks, crustaceans, arachnids, echinoderms andinsects), plants, fungi (e.g., but not limited to mushrooms, mold, andyeast), and microorganisms (e.g., but not limited to bacteria andviruses) in view of the specification and examples provided herein.Further, a skilled artisan can adapt methods of uses described hereinfor various applications of different tissue-mimic devices.

In accordance with some embodiments of the invention, the devicesdescribed herein can be used to mimic function of a blood-brain barrier.For example, brain cells (e.g., neurons, and/or astrocytes) can becultured on one surface of the membrane and blood vessel-associatedcells (e.g., endothelial cells, fibroblasts, smooth muscle cells,pericytes, and/or any combinations thereof) on another surface of themembrane. It is commonly believed that the native brain cells areusually exposed to a high shear stress. Accordingly, in someembodiments, application of a mechanical strain/stress to the braincells can be used instead in place of a high-shear flow.

Methods of Making a Device Described Herein

Details on how the device 200 is formed will now be discussed inaccordance with an embodiment. Embodiments of various devices describedherein enables us to leverage the control of microfluidic technology andreconstitute the organ level function associated with the primary celltype, e.g., breathing/strain of airway epithelial cells while for thefirst time offering a reduced stress environment and increased overheadspace for growth, only feasible in a larger meso-scale channel. In someembodiments, two technologies are used to fabricate the devicesdescribed herein. The bottom microchannel, which, in one embodiment, isapproximately 100 μm tall, can be manufactured using any conventionalfabrication methods, including, e.g., injection molding, embossing,etching, casting, machining, stamping, lamination, photolithography, orany combinations thereof. In one embodiment, the bottom microchannel ismanufactured by a process comprising traditional photolithography, atechnique useful for creating fluidic features of the order ten toseveral hundred microns. As seen in FIG. 3A, the end result ofphotolithography is a silicon wafer with the design of microchannelsraised to a pre-determined height (e.g., about 25 μm to about 1000 μm).In one embodiment, the bottom microchannel is manufactured by a processcomprising soft lithography techniques, the details of which aredescribed in “Soft Lithography in Biology and Biochemistry,” byWhitesides, et al., published Annual Review, Biomed Engineering,3.335-3.373 (2001), as well as “An Ultra-Thin PDMS Membrane As ABio/Micro-Nano Interface: Fabrication And Characterization”, byThangawng et al., Biomed Microdevices, vol. 9, num. 4, 2007, p. 587-95,both of which are hereby incorporated by reference. After the wafer withthe design of raised features is made, a curable biocompatible polymer,e.g., but not limited to, PDMS, polyurethane, SEBS, polypropylene, andany combinations thereof, can be casted into the mold, which then formsthe microchannel. In some embodiments, the bottom microchannel can befabricated using a combination of two or more techniques describedherein.

The top mesochannel, which, in one embodiment, is approximately 1 mmtall, can be can be manufactured using any conventional fabricationmethods, including, e.g., injection molding, embossing, etching,casting, machining, stamping, lamination, photolithography, or anycombinations thereof. In some embodiments, the top mesochannel can beless desirable to be made using photolithography because SU-8 structuresof larger size (e.g., in millimeter range) can have material defects. Insome embodiments, the solid free-form fabrication technology such asstereo-lithography can be used to make a mold for the mesochannel, dueto its superior surface finish and resolution. Generally,stereo-lithography can be used to make a mold with features having aminimum dimension of at least about 20 μm to about 50 μm, depending onthe machine. An example schematic diagram of the stereo-lithographyprocedure is shown in FIG. 3B. In this process, the silicon wafer usedin photolithography is being replaced by a layer of solidified resin. Asthe process continues, the features of the channels are etched by thelaser and thus solidified, and the result is a wafer-like device madeentirely of thermoplastic resin.

The design of the device described herein can be drawn in 3D CAD designsoftware, e.g., SolidWorks, which is then read by a stereolithographymachine and drawn in thermoset resin with an ultraviolet laser. Thedrawing and final mold are shown in FIG. 3C.

In some embodiments where the height of the operating channel(s) is muchsmaller than the height of the mesochannel (e.g., by a factor of 2 orhigher, such as a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher), theinventors have surprisingly discovered that stereo-lithography can beused to produce a mold with features of different scales (e.g.,mesochannel vs. operating channels).

Similar to the silicon wafer with microstructures, which PDMS is thencast upon, the PDMS is cast into the entire thermoplastic mold whichthen forms the mesochannels. In addition to PDMS, other materials suchas polyurethanes (e.g., see PCT/US12/36920 for use of polyurethanematerials to produce microfluidic devices),styrene-ethylene-butylene-styrene (SEBS) (e.g., see, US2011/0085949 foruse of thermoplastic elastomers to produce microfluidic devices),polypropylene, silicon, or any combinations thereof. The content of thepatent applications are incorporated herein by reference.

Without wishing to be limiting, in some embodiments, the devicesdescribed herein can be produced as a monolithic device or as individualcomponents (e.g., a first portion of the body comprising a mesochannel,a second portion of the body comprising a microchannel, and a membrane),which can then be assembled together to form a device described herein.Each individual component can be produced by a conventionalmanufacturing method such as injection molding, extrusion, casting,lamination, embossing, compression molding, solvent casting, an additivemanufacturing method (e.g., 3D printing), or any combinations thereof.

The top outer body portion 204 can have a thickness of any dimension,depending, in part, on the height of the mesochannel 250A. In someembodiments, the thickness of the top outer body portion 204 can beabout 1 mm to about 100 mm, or about 2 mm to about 75 mm, or about 3 mmto about 50 mm, or about 3 mm to about 25 mm. In one embodiment, thethickness of the top outer body portion 204 can be about 4.8 mm.

The bottom outer body portion 206 can have a thickness of any dimension,depending, in part, on the height of the microchannel 250B. In someembodiments, the thickness of the bottom outer body portion 206 can beabout 50 μm to about 10 mm, or about 75 μm to about 8 mm, or about 100μm to about 5 mm, or about 200 μm to about 2.5 mm. In one embodiment,the thickness of the bottom outer body portion 206 can be about 1 mm toabout 1.5 mm. In one embodiment, the thickness of the bottom outer bodyportion 206 can be about 0.2 mm to about 0.5 mm.

Once the top and bottom outer body portions 204, 206 are formed andremoved from their respective molds, the access ports can be made toaccess the channels. In one embodiment, the ports are created using a0.5 mm biopsy punch at 90° (FIG. 2C, top panels). This sharp redirectionof flow can create forces that can cause undesirable effects on thecells, local pressure variations and cell aggregation. An alternativedesign is for the punch to be used at a lower angle (FIG. 2C, bottompanels). This can mitigate the problems associated with the purelyvertical punch. In this embodiment, the access ports (e.g., for inletsand outlets) are positioned on the lateral sides of the devicesdescribed herein such that the inlet channels and outlet channels can beangled at an angle smaller than 90 degrees, e.g., ranging from about 15degrees to about 45 degrees. In one embodiment, the inlet channels andoutlet channels can be angled at an angle of about 25 degrees.

The membrane 208 can be engineered for a variety of purposes, somediscussed above. For example, the pores on the membrane 208 can becoated or filled with ECM molecules or gels, such as MATRIGEL, laminin,collagen, fibronectin, fibrin, elastin, etc., which are known to thoseskilled in the art. The tissue-tissue interface can be coated byculturing different types of cells on each side of the membrane 208, asshown in FIG. 2D. In particular, as shown in FIG. 2D, one type of cellsare coated on one side of the membrane 208 whereas another type of cellsare coated on the opposing side of the membrane 208.

As described earlier, the membrane 208 can be rigid or flexible. In someembodiments, the membrane 208 can be rigid, e.g., a polycarbonate orpolyester membrane. In some embodiments, the membrane 208 can beflexible, e.g., a PDMS membrane.

In general, the membrane 208 is sandwiched between the top outer bodyportion 204 comprising a mesochannel 250A and the bottom outer bodyportion 206 comprising a microchannel 250B, whereby the channel walls234, 244 as well as the outside walls 238, 248 are aligned usingappropriate manufacturing equipment and techniques. Thereafter, themembrane 208 is fastened to the channel walls 234, 244 and optionaloutside walls 238, 248 using an appropriate adhesive or epoxy, physicalclamping and/or plasma bond between the two PDMS surfaces, in order toform a fluidic seal between the membrane and the top body portion 204and the bottom body portion 206.

For example, a fluidic seal can be formed by a chemical bond between themembrane and the top body portion and the bottom body portion. In oneembodiment, the chemical bond can be created with an adhesive chemicalcoating, e.g., 3-Aminopropyl-triethoxysilane (APTES), which can createan irreversible bond between the polycarbonate or polyester membrane,and the top body portion and the bottom body portion. See, e.g., Aran etal. “Irreversible, direct bonding of nanoporous polymer membranes toPDMS or glass microdevices.” Lab Chip. 2010 Mar. 7; 10(5):548-52, foruse of 3-aminopropyltriethoxysilane as a chemical crosslinking agent tointegrate polymer membranes such as polyethersulfone and polyethyleneterephthalate, with PDMS and glass microfluidic channels. The APTESprocedure is described in FIG. 4A.

In some embodiments, a fluidic seal can be formed by clamping themembrane between the top and bottom body portions 204, 206 (e.g., PDMSportions) utilizing all membrane-PDMS surface area. Clamping themembrane between the top and bottom body portions 204, 206 can beachieved by placing the device between two plates, e.g., acrylic plates.The plates (e.g., acrylic plates) can then be clamped together eitherwith screws or clips as seen in FIG. 4B. This method can allow the userto access the membrane after the experiment with minimal damage to thecells on the membrane, yielding a higher quality images.

In some embodiments where the top and bottom body portions are made ofPDMS, a fluidic seal can be formed by cutting a membrane smaller thanthe PDMS surface area and plasma bonding the PDMS together around themembrane, e.g., as shown in FIG. 4C. This method can allow the user toaccess the membrane after the experiment with minimal damage to thecells on the membrane, yielding a higher quality images.

FIG. 20 illustrates a schematic of a system having multiple devices inaccordance with an embodiment. In particular, as shown in FIG. 20, thesystem 700 includes one or more CPUs 702 coupled to one or more fluidsources 704 and external force sources (e.g., pressure sources) (notshown), whereby the preceding are coupled to three devices 706A, 706B,and 706C. It should be noted that although three devices 706 are shownin this embodiment, fewer or greater than three devices 706 can be used.In the system 700, two of the three devices (i.e. 706A and 706B) areconnected in parallel with respect to the fluid source 704 and devices706A and 706C are connected in serial fashion with respect to the fluidsource 704. It should be noted that the shown configuration is only oneexample and any other types of connection patterns can be utilizeddepending on the application. In some embodiments, a system can be theone described in the International Patent Application No.PCT/US12/68725, entitled “Integrated human organ-on-chipmicrophysiological systems,” where one or more devices described hereincan be fluidically connected to form the system. For example, as shownin FIG. 19, about 8-16 devices described herein can be fluidicallyconnected to form one or more systems maintained in an incubator.

In the example shown, fluid from the fluid source 704 is provideddirectly to the fluid inlets of devices 706A and 706B. As the fluidpasses through device 706A, it is output directly into the fluid inletport of devices 706B and 706C. Additionally, the fluid outlet fromdevice 706B is combined with the output from device 706A into device706C. With multiple devices operating, it is possible to monitor, usingsensor data, how the cells in the fluid or membrane behave after thefluid has been passed through another controlled environment. Thissystem thus allows multiple independent “stages” to be set up, wherecell behavior in each stage can be monitored under simulatedphysiological conditions and controlled using the devices 706. One ormore devices are connected serially can provide use in studying chemicalcommunication between cells. For example, one cell type can secreteprotein A in response to being exposed to a particular fluid, wherebythe fluid, containing the secreted protein A, exits one device and thenis exposed to another cell type specifically patterned in anotherdevice, whereby the interaction of the fluid with protein A with theother cells in the other device can be monitored (e.g. paracrinesignaling). For the parallel configuration, one or more devicesconnected in parallel can be advantageous in increasing the efficiencyof analyzing cell behavior across multiple devices at once instead ofanalyzing the cell behavior through individual devices separately.

Additional Examples of Cytokines

As used herein, the term “cytokine” refers to an agent that canstimulate, inhibit, and/or mediate a cellular process, including, e.g.,but not limited to, proliferation, differentiation, inflammation,apoptosis, cellular metabolism, cytoskeletal regulation, cell adhesion,cell migration, angiogenesis, DNA repair, protein synthesis, and anycombinations thereof. A “cytokine” can be or include a small molecule, abiological molecule (e.g., but not limited to, a protein, peptide,nucleic acid, lipid, carbohydrate, glycoprotein, glycolipid,proteoglycan, lipoprotein), an antibody, oligonucleotide, a metal, avitamin, or any combinations thereof. For example, a cytokine caninclude, but are not limited to, a growth-promoting agent, a celldifferentiation agent, an anti-inflammatory agent, a pro-inflammatoryagent, an apoptosis-inducing agent, an anti-apoptotic agent, apro-angiogenic agent, an anti-angiogenic agent, or any combinationsthereof.

In some embodiments, the cytokine can include a pro-inflammatory agent.As used herein, the term “pro-inflammatory agent” refers to an agentthat can directly or indirectly induce or mediate an inflammatoryresponse in cells, or is directly or indirectly involved in productionof a mediator of inflammation. A variety of proinflammatory agents areknown to those skilled in the art. Illustratively, pro-inflammatoryagents include, without limitation, eicosanoids such as, for example,prostaglandins (e.g., PGE2) and leukotrienes (e.g., LTB4); gases (e.g.,nitric oxide (NO)); enzymes (e.g., phospholipases, inducible nitricoxide synthase (iNOS), COX-1 and COX-2); and cytokines such as, forexample, interleukins (e.g., IL-1α, IL-β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-8, IL-10, IL-12 and IL-18), members of the tumor necrosis factorfamily (e.g., TNF-α, TNF-β and lymphotoxin β), interferons (e.g., IFN-βand IFN-γ), granulocyte/macrophage colony-stimulating factor (GM-CSF),transforming growth factors (e.g., TGF-β1, TGF-β2 and TGF-β3, leukemiainhibitory factor (LTF), ciliary neurotrophic factor (CNTF), migrationinhibitory factor (MTF), monocyte chemoattractant protein (MCP-I),macrophage inflammatory proteins (e.g., MIP-1α, MIP-1β and MIP-2), andRANTES, as well as environmental or physical agents such as silicamicro- and nano-particles and pathogens. In some embodiments, at leastone or more of these pro-inflammatory agents can be added to a cellculture medium, e.g., to stimulate or challenge tissue-specific cellsand/or immune cells within the device to simulate an inflammatoryresponse or an inflammation-associated disease, disorder, or injury invivo.

In some embodiments, the cytokine can include an anti-inflammatoryagent. The term “anti-inflammatory agent,” as used herein, refers to anagent capable of counteracting the effects of pro-inflammatory and/orinflammatory agents and other agents that mediate an inflammatorycondition or reaction. Examples of an anti-inflammatory agent caninclude, but are not limited to, inhibitors of any pro-inflammatoryagents as described above, e.g., in a form of soluble receptors,receptor antagonists, aptamers, antibodies, or any combinations thereof;and/or an agent that can mediate an inflammatory pathway in a cell,e.g., in a form of soluble proteins, antisense oligonucleotides, siRNA,shRNA, vectors, or any combinations thereof. For example, ananti-inflammatory agent can include an agent that can inhibit aparticular protein function and/or silence a specific gene that inducesinflammation; or an agent that can promote a particular protein functionand/or express a specific gene that inhibits inflammation. In someembodiments, an anti-inflammatory agent can be or include a steroid, anonsteroidal anti-inflammatory drug, an analgesic, an inhibitor of atleast one or more chemokines (e.g., but not limited to, CXCL-8, CCL2,CCL3, CCL4, CCL5, CCL11, and CXCL10) and/or a COX-2 inhibitor. A varietyof anti-inflammatory agents are known to those skilled in the art, e.g.,as described in International Patent App. NO. WO 2004/082588, thecontent of which is incorporated herein by reference, and can be addedto a cell culture medium and/or used to stimulate or challengetissue-specific cells and/or immune cells within the device to provokean anti-inflammatory response.

In some embodiments, the cytokine can include a growth-promoting agent.As used herein, the term “growth-promoting agent” refers to an agentthat stimulates cell proliferation. Examples of a growth-promoting agentcan include but are not limited to any art-recognized growth factorssuch as Bone morphogenetic proteins (BMPs); Brain-derived neurotrophicfactor (BDNF); Epidermal growth factor (EGF); Erythropoietin (EPO);Fibroblast growth factor (FGF); Glial cell line-derived neurotrophicfactor (GDNF); Granulocyte colony-stimulating factor (G-CSF);Granulocyte macrophage colony-stimulating factor (GM-CSF); Hepatocytegrowth factor (HGF); Hepatoma-derived growth factor (HDGF); Insulin-likegrowth factor (IGF); Myostatin (GDF-8); Nerve growth factor (NGF) andother neurotrophins; Platelet-derived growth factor (PDGF);Thrombopoietin (TPO); Transforming growth factor alpha (TGF-α);Transforming growth factor beta (TGF-β); Vascular endothelial growthfactor (VEGF); Placental growth factor (PlGF); hormones, steroidhormones, and any combinations thereof.

In some embodiments, the cytokine can include a differentiation agent asdescribed earlier. Appropriate differentiation agent(s) can be selectedbased on different cell types, including, e.g., stem cells, andundifferentiated or partially differentiated cells.

In some embodiments, the cytokine can include an apoptosis modulatingagent. The term “apoptosis modulating agents,” as used herein, refers toagents which are involved in modulating (e.g., inhibiting, decreasing,increasing, promoting) apoptosis. Apoptosis is generally known as aprocess of programmed cell death. Examples of apoptosis modulatingagents include, but are not limited to, Fas/CD95, TRAMP, TNF RI, DR1,DR2, DR3, DR4, DR5, DR6, FADD, RIP, TNFα, Fas ligand, antibodies toFas/CD95 and other TNF family receptors, TRAIL, antibodies to TRAIL-R1or TRAIL-R2, Bcl-2, p53, BAX, BID, BAD, BAK, Akt, CAD, PI3 kinase, PPl,and caspase proteins. Modulating agents broadly include agonists andantagonists of TNF family receptors and TNF family ligands. Apoptosismodulating agents can be soluble or membrane bound (e.g. ligand orreceptor).

In some embodiments, the cytokine can include a pro-angiogenic agent. Asused herein, the term “pro-angiogenic agent” is intended to mean anagent that directly or indirectly stimulates, enhances and/or stabilizesangiogenesis. Exemplary pro-angiogenic agents include, but are notlimited to, VEGF, FGF, Ang1, Ang2, PDGF-BB, and any combinationsthereof.

In some embodiments, the cytokine can include an anti-angiogenic agent.As used herein, the term “anti-angiogenic agent” refers to an agent thatdirectly or indirectly reduces or inhibits formation of new bloodvessels, and/or destabilizes the formed blood vessels. Examples ofanti-angiogenic agents include, but are not limited to, inhibitorsand/or antagonists of the pro-angiogenic agents as described above,soluble VEGF receptors, angiopoietin 2, TSP-1, TSP-2, angiostatin,endostatin, vasostatin, platelet factor-4, and any combinations thereof.

Embodiments of Various Aspects Described Herein can be Defined in any ofthe Following Numbered Paragraphs:

-   1. A device comprising:    -   a. a body comprising a central channel therein; and    -   b. a membrane positioned within the central channel and along a        plane, the membrane configured to separate the central channel        to form at least one microchannel and at least one mesochannel,        wherein the height of the mesochannel is substantially greater        than the height of the microchannel.-   2. The device of paragraph 1, wherein the height ratio of the    mesochannel to the microchannel ranges from about 2.5:1 to about    50:1.-   3. The device of paragraph 1 or 2, wherein the height ratio of the    mesochannel to the microchannel ranges from about 5:1 to about 25:1.-   4. The device of any of paragraphs 1-3, wherein the membrane is    rigid.-   5. The device of any of paragraphs 1-3, wherein the membrane is at    least partially flexible.-   6. The device of any of paragraphs 1-5, wherein the membrane has a    thickness of about 1 μm to about 100 μm.-   7. The device of any of paragraphs 1-5, wherein the membrane has a    thickness of about 100 nm to about 50 μm.-   8. The device of any of paragraphs 1-7, wherein the membrane is    non-porous.-   9. The device of any of paragraphs 1-8, wherein the membrane is at    least partially porous.-   10. The device of paragraph 9, wherein pores of the membrane has a    diameter of about 0.1 μm to about 15 μm.-   11. The device of paragraph 9 or 10, wherein center-to-center pore    spacing ranges from about 1 μm to about 100 μm.-   12. The device of any of paragraphs 1-11, wherein one end of the    mesochannel is adapted to engage to a gas-flow modulation device.-   13. The device of paragraph 12, wherein the gas-flow modulation    device is adapted to provide a uni-directional or bi-directional    flow of gas.-   14. The device of paragraph 12 or 13, wherein the gas-flow    modulation device comprises a gas-receiving chamber having at least    one end enclosed by a flexible diaphragm.-   15. The device of paragraph 14, wherein the gas-receiving chamber    expands or contracts as the flexible diaphragm moves.-   16. The device of any of paragraphs 1-15, wherein another end of the    mesochannel is adapted to engage to a gas-flow generator.-   17. The device of any of paragraphs 1-16, wherein the body is    further adapted to provide mechanical modulation of the membrane    within the central channel.-   18. The device of paragraph 17, wherein the body further comprises a    first operating channel separated from the microchannel and the    mesochannel by a first channel wall, wherein a first edge of the    membrane is fastened to the first channel wall and a second edge of    the membrane is fastened to an opposite wall of the central channel;    and wherein the first operating channel is positioned around the    membrane such that a pressure differential applied between the first    operating channel and the central channel causes the membrane to    stretch or retract in a first desired direction along the plane    within the central channel.-   19. The device of paragraph 18, wherein the body further comprises a    second operating channel separated from the microchannel and the    mesochannel by a second channel wall, wherein the second edge of the    membrane is fastened to the second channel wall; and wherein the    second operating channel is positioned around the membrane such that    the pressure differential applied between the second operating    channel and the central channel causes the membrane to stretch or    retract in a second desired direction along the plane within the    central channel.-   20. The device of paragraph 18 or 19, wherein a first height of the    first operating channel and a second height of the second operating    channel are smaller than the height of the central channel.-   21. The device of any of paragraphs 18-20, wherein at least one or    both of the first operating channel and the second operating channel    are symmetrically arranged around the membrane.-   22. The device of any of paragraphs 1-21, wherein the height of the    microchannel ranges from about 20 μm to about 1 mm.-   23. The device of any of paragraphs 1-21, wherein the height of the    microchannel ranges from about 50 μm to about 200 μm.-   24. The device of any of paragraphs 1-23, wherein the dimensions of    the mesochannel are configured to provide a fluid shear stress    appropriate for cell growth and/or cell differentiation.-   25. The device of any of paragraphs 1-24, wherein the height of the    mesochannel is sufficient for formation of a stratified or    three-dimensional tissue.-   26. The device of any of paragraphs 1-25, wherein an aspect ratio of    the height of the mesochannel to the width of the central channel    ranges from about 1:5 to about 25:1.-   27. The device of any of paragraphs 1-26, wherein the height of the    mesochannel ranges from about 100 μm to about 50 mm.-   28. The device of any of paragraphs 1-27, wherein the width of the    central channel ranges from about 200 μm to about 10 mm.-   29. The device of any of paragraphs 1-28, wherein at least one    surface of the membrane comprises cells adhered thereto.-   30. The device of paragraph 29, wherein the cells form one or more    cell layers.-   31. The device of paragraph 29 or 30, wherein the cells are selected    from the group consisting of mammalian cells, plant cells, insect    cells, and any combinations thereof-   32. The device of paragraph 31, wherein the mammalian cells comprise    human cells.-   33. The device of paragraph 31, wherein the mammalian cells comprise    animal cells.-   34. The device of any of paragraphs 29-33, wherein the cells display    at least one characteristic corresponding to a pre-determined    physiological endpoint.-   35. The device of paragraph 34, wherein the pre-determined    physiological endpoint is selected from the group consisting of a    mature state, a differentiated state, a precursor state, a    stratified state, a pseudo-stratified state, a confluency state, an    inflamed state, an infected state, a stimulated state, an activated    state, an inhibitory state, a normal healthy state, a    disease-specific state, a growth state, a migratory state, a    metamorphosing state, or any combinations thereof-   36. The device of paragraph 35, wherein the disease-specific state    is a specific stage of a disease, disorder or injury.-   37. The device of paragraph 35 or 36, wherein the disease-specific    state comprises a cancerous state.-   38. The device of any of paragraphs 35-37, wherein the    disease-specific state is associated with an intestinal disease    selected from the group consisting of inflammatory bowel disease,    Crohn's disease, ulcerative colitis, celiac disease, angiodysplasia,    appendicitis, bowel twist, chronic functional abdominal pain,    coeliac disease, colorectal cancer, diverticular disease,    endometriosis, enteroviruses, gastroenteritis, Hirschsprung's    disease, ileitis, irritable bowel syndrome, polyp, pseudomembranous    colitis, or any combinations thereof.-   39. The device of any of paragraphs 35-37, wherein the    disease-specific state is associated with a lung disease selected    from the group consisting of asthma, chronic obstructive pulmonary    disease (COPD), pulmonary hypertension, radiation induced injury,    cystic fibrosis, or any combination thereof.-   40. The device of any of paragraphs 35-37, wherein the    disease-specific state is associated with an airborne disease.-   41. The device of paragraph 40, wherein the airborne disease is a    bacterial infection or a viral infection.-   42. The device of any of paragraphs 29-41, wherein at least a    portion of the cells are selected from the group consisting of    epithelial cells, endothelial cells, fibroblasts, smooth muscle    cells, basal cells, ciliated cells, mucus-secreting cells, columnar    cells, goblet cells, muscle cells, immune cells, neural cells,    hematopoietic cells, lung cells (e.g., alveolar epithelial cells,    airway cells, bronchial cells, tracheal cells, and nasal epithelial    cells), gut cells, intestinal cells, brain cells, stem cells, skin    cells, liver cells, heart cells, spleen cells, kidney cells,    pancreatic cells, reproductive cells, blood cells (including, e.g.,    white blood cells, red blood cells, platelets, and hematopoietic    stem cells and progenitor cells) and any combinations thereof-   43. The device of any of paragraphs 29-42, wherein the cells are    selected to create an in vitro model that mimics cell behavior of at    least a portion of a tissue.-   44. The device of paragraph 43, wherein the tissue is selected from    the group consisting of airway, bronchus, gut, skin, choroid plexus,    liver, heart, and gastrointestinal tract.-   45. The device of any of paragraphs 29-44, wherein a first surface    of the membrane facing the mesochannel comprises tissue-specific    cells requiring low shear and/or space to form a stratified tissue.-   46. The device of paragraph 45, wherein the tissue-specific cells    comprise epithelial cells, basal cells, ciliated cells, columnar    cells, goblet cells, fibroblasts, smooth muscle cells, or any    combinations thereof-   47. The device of paragraph 45 or 46, wherein the tissue-specific    cells selected to create the in vitro model that mimics cell    behavior of at least a portion of an airway comprises airway    epithelial cells, bronchial epithelial cells, nasal epithelial    cells, or any combinations thereof.-   48. The device of any of paragraphs 29-47, wherein a second surface    of the membrane facing the microchannel comprises blood    vessel-associated cells.-   49. The device of paragraph 48, wherein the blood vessel-associated    cells comprise endothelial cells, fibroblasts, smooth muscle cells,    pericytes, or any combinations thereof-   50. The device of any of paragraphs 1-49, wherein the membrane is    coated with at least one cell adhesion agent.-   51. The device of paragraph 50, wherein said at least one cell    adhesion agent comprises an extracellular matrix molecule.-   52. The device of paragraph 51, wherein the extracellular matrix    molecule comprises glycoproteins, collagen, fibronectin, laminin,    vitronectin, elastins, fibrin, proteoglycans, heparin sulfate,    chondroitin sulfate, keratan sulfate, hyaluronic acid, fibroin,    chitosan, or any combinations thereof-   53. The device of any of paragraphs 1-52, wherein the body of the    device and/or the membrane comprises a biocompatible polymer.-   54. The device of paragraph 53, wherein the biocompatible polymer    comprises polydimethylsiloxane (PDMS), polyurethane,    styrene-ethylene-butylene-styrene (SEBS), polypropylene,    polycarbonate, polyester, polypropylene, silicon, or any    combinations thereof.-   55. The device of any of paragraphs 1-54, wherein the body of the    device and/or the membrane comprises an extracellular matrix    polymer, gel, or scaffold.-   56. The device of any of paragraphs 1-55, wherein the central    channel is linear.-   57. The device of any of paragraphs 1-55, wherein the central    channel comprise a non-linear portion.-   58. The device of any of paragraphs 1-57, wherein the height of the    first and/or second operating channel is larger than the height of    the central channel.-   59. The device of any of paragraphs 1-57, wherein the height of the    first and/or second operating channels is substantially the same as    or smaller than the height of the central channel.-   60. A method comprising:    -   providing at least one device comprising:        -   a. a body comprising a central channel therein; and        -   b. an at least partially porous membrane positioned within            the central channel and along a plane, the membrane            configured to separate the central channel to form a first            sub-channel and a second sub-channel, wherein at least the            first sub-channel has a height sufficient to form a            stratified structure;    -   seeding tissue-specific cells on a first surface of the membrane        facing the first sub-channel; and    -   culturing the tissue-specific cells on the first surface at a        gas-liquid interface.-   61. The method of paragraph 60, wherein the tissue-specific cells    form a first cell monolayer prior to said culturing at the    gas-liquid interface.-   62. The method of paragraph 61, wherein the first cell monolayer is    formed by culturing the tissue-specific cells submerged in a first    liquid fluid within the first sub-channel.-   63. The method of any of paragraphs 60-62, wherein the gas-liquid    interface is formed by having a gaseous fluid in the first    sub-channel and a second liquid fluid in the second sub-channel.-   64. The method of paragraph 63, wherein the second liquid fluid    comprises at least one differentiation-inducing agent.-   65. The method of any of paragraphs 60-64, wherein at least a    portion of the tissue-specific cells reach a pre-determined    physiological endpoint upon said culturing at the gas-liquid    interface for a period of time.-   66. The method of any of paragraphs 60-65, further comprising    flowing a gaseous fluid through the first sub-channel.-   67. The method of any of paragraphs 60-66, further comprising    flowing a liquid fluid through the second sub-channel.-   68. A method comprising:    -   providing at least one device comprising:        -   a. a body comprising a central channel therein; and        -   b. an at least partially porous membrane positioned within            the central channel and along a plane, the membrane            configured to separate the central channel to form a first            sub-channel and a second sub-channel, wherein at least the            first sub-channel has a height sufficient to form a            stratified structure; and        -   c. tissue-specific cells on a first surface of the membrane            facing the first sub-channel, wherein the cells display at            least one characteristic corresponding to a pre-determined            physiological endpoint.        -   flowing a gaseous fluid through the first sub-channel; and        -   flowing a liquid fluid through the second sub-channel.-   69. The method of any of paragraphs 66-68, wherein the gaseous fluid    is maintained at a static flow.-   70. The method of any of paragraphs 66-68, wherein the gaseous fluid    is continuously flowed through the first sub-channel.-   71. The method of any of paragraphs 66-68, wherein the gaseous fluid    is intermittently or cyclically flowed through the first    sub-channel.-   72. The method of any of paragraphs 60-71, wherein the height of the    first sub-channel is configured to provide an air shear stress    appropriate for cell growth and/or cell differentiation.-   73. The method of paragraph 72, wherein the air shear stress ranges    from about 0.01 dynes/cm² to about 2000 dynes/cm².-   74. The method of any of paragraphs 60-73, wherein the height of the    first sub-channel is at least about 100 μm or about 500 μm.-   75. The method of any of paragraphs 60-74, wherein the second    sub-channel has a height that is substantially smaller than the    height of the first sub-channel.-   76. The method of paragraph 75, wherein the second sub-channel has a    height of about 20 μm to about 1 mm or about 50 μm to about 200 μm.-   77. The method of any of paragraphs 60-76, wherein the height of the    second sub-channel is substantially same as the height of the first    sub-channel.-   78. The method of any of paragraphs 60-77, wherein the    pre-determined physiological endpoint is selected from the group    consisting of a mature state, a differentiated state, a precursor    state, a stratified state, a pseudo-stratified state, a confluency    state, an inflamed states, an infected state, a stimulated state, an    activated state, an inhibitory state, a normal healthy state, a    disease-specific state, a growth state, a migratory state, a    metamorphosing state, or any combinations thereof-   79. The method of paragraph 78, wherein the disease-specific state    is a specific stage of a disease, disorder or injury.-   80. The method of paragraph 78 or 79, wherein the disease-specific    state comprises a cancerous state.-   81. The method of any of paragraphs 65-80, wherein the    pre-determined physiological endpoint is detected by the presence of    at least one marker associated with the pre-determined physiological    endpoint.-   82. The method of any of paragraphs 60-81, wherein the    tissue-specific cells comprise mammalian cells.-   83. The method of any of paragraphs 60-82, wherein the    tissue-specific cells comprise airway, bronchial, and/or nasal    epithelial cells.-   84. The method of paragraph 83, wherein the physiological endpoint    of the airway or bronchial epithelial cells is differentiation of    the airway or bronchial epithelial cells to ciliated cells and/or    mucus-secreting cells.-   85. The method of paragraph 84, wherein the differentiated state is    detected by the presence of at least one of the cilia-associated    markers, goblet cell-associated markers, and tight    junction-associated markers.-   86. The method of any of paragraphs 60-85, further comprising    treating differentiated cells with retinoic acid.-   87. The method of paragraph 86, wherein the retinoic acid reverses    squamous phenotype.-   88. The method of any of paragraphs 60-87, wherein one end of the    first sub-channel is adapted to engage to a gas-flow modulation    device.-   89. The method of paragraph 88, wherein gas-flow modulation device    is adapted to provide a unidirectional and/or a bidirectional flow    of the gaseous fluid.-   90. The method of paragraph 89, wherein the bidirectional flow of    the gaseous fluid simulates air flow during respiration.-   91. The method of any of paragraphs 88-90, wherein the gas-flow    modulation device comprises a gas-receiving chamber having at least    one end enclosed by a flexible diaphragm.-   92. The method of paragraph 91, wherein the gas-receiving chamber    expands or contracts as the flexible diaphragm moves.-   93. The method of any of paragraphs 60-92, further comprising    determining ciliary clearance of a particle flowing through the    first sub-channel.-   94. The method of any of paragraphs 60-93, further comprising    forming a second cell layer on a second surface of the membrane    facing the second sub-channel.-   95. The method of paragraph 94, wherein the second cell layer    comprises blood vessel-associated cells.-   96. The method of paragraph 95, wherein the blood vessel-associated    cells comprise endothelial cells, fibroblasts, smooth muscle cells,    pericytes, or any combinations thereof-   97. The method of any of paragraphs 60-96, further comprising    creating within the central channel an in vitro model that mimics a    tissue-specific condition (e.g., in a normal healthy state or in a    disease-specific state).-   98. The method of paragraph 97, wherein the tissue-specific cells    are adapted to display at least one characteristic associated with    the tissue-specific condition in a disease-specific state.-   99. The method of paragraph 98, wherein the tissue-specific cells    are disease-specific cells isolated from at least one subject.-   100. The method of paragraph 98, wherein the tissue-specific cells    are contacted with a condition-inducing agent that is capable of    inducing the tissue-specific cells to acquire at least one    characteristic associated with the disease-specific state.-   101. The method of paragraph 100, wherein the condition-inducing    agent comprises a physical agent or an environmental stimulus (e.g.,    radiation or air flow rhythm).-   102. The method of paragraph 100 or 101, wherein the    condition-inducing agent comprises a chemical and/or biological    agent (e.g., pathogens, and/or pro-inflammatory agents).-   103. The method of any of paragraphs 97-102, wherein the    tissue-specific condition is associated with a lung disease,    disorder and/or injury or an airborne disease.-   104. The method of paragraph 103, wherein the tissue-specific cells    selected to mimic the condition associated with the lung disease,    disorder and/or injury or the airborne disease comprise airway    epithelial cells, bronchial epithelial cells, and/or nasal    epithelial cells.-   105. The method of paragraph 103 or 104, wherein the lung disease,    disorder and/or injury is selected from the group consisting of    acute lung injuries, chronic lung disorders, lung infections, and    lung cancer.-   106. The method of paragraph 103, wherein the airborne disease is a    viral infection or a bacterial infection.-   107. The method of paragraph 105, wherein the acute lung injuries    comprise lung injuries resulting from bacterial sepsis, hemorrhagic    shock, toxic inhalation, a drug-induced lung injury (e.g.,    bleomycin-induced lung injury), or any combinations thereof-   108. The method of paragraph 105, wherein the chronic lung disorders    comprises chronic obstructive pulmonary disorder (COPD), asthma,    cystic fibrosis, fibrotic conditions, sarcoidosis, idiopathic lung    fibrosis.-   109. The method of any of paragraphs 97-102, wherein the    tissue-specific condition is associated with an intestinal disease    or disorder.-   110. The method of paragraph 109, wherein the tissue-specific cells    selected to mimic the condition associated with the intestinal    disease or disorder comprise intestinal cells, colon cells, appendix    cells, ileum cells, caecum cells, duodenum cells, jejunum cells, or    any combinations thereof-   111. The method of paragraph 109 or 110, wherein the intestinal    disease, disorder and/or injury is selected from the group    consisting of inflammatory bowel disease, Crohn's disease,    ulcerative colitis, celiac disease, angiodysplasia, appendicitis,    bowel twist, chronic functional abdominal pain, coeliac disease,    colorectal cancer, diverticular disease, endometriosis,    enteroviruses, gastroenteritis, Hirschsprung's disease, ileitis,    irritable bowel syndrome, polyp, pseudomembranous colitis, or any    combinations thereof.-   112. The method of any of paragraphs 60-111, further comprising    contacting the tissue-specific cells with a test agent.-   113. The method of paragraph 112, wherein the tissue-specific cells    are contacted with the test agent by delivery as an aerosol or    liquid through the first sub-channel and/or via diffusion from the    second sub-channel.-   114. The method of paragraph 112 or 113, wherein the test agent is    selected from the group consisting of proteins, peptides, nucleic    acids, antigens, nanoparticles, environmental toxins or pollutant,    cigarette smoke, chemicals or particles used in cosmetic products,    small molecules, drugs or drug candidates, vaccine or vaccine    candidates, aerosols, pro-inflammatory agents, naturally occurring    particles including pollen, chemical weapons, viruses, bacteria,    unicellular organisms, cytokines, and any combinations thereof.-   115. The method of any of paragraphs 112-114, further comprising    performing a pharmacokinetic, a pharmacodynamics, or a    pharmacokinetic-pharmacodynamic (PK-PD) assay and/or analysis of an    effect of the test agent on the cells, thereby determining an in    vitro pharmacokinetic and/or pharmacodynamics effect of the test    agent on the cells.-   116. The method of any of paragraphs 112-115, further comprising    measuring response of the cells on at least one side of the membrane    to the test agent, the gaseous fluid exiting the first sub-channel,    the liquid fluid exiting the second sub-channel, or any combinations    thereof.-   117. The method of paragraph 116, wherein said measuring the    response of the cells comprises measuring adhesion of immune cells    that are flowing through the second sub-channel, cell labeling,    immunostaining, optical or microscopic imaging (e.g.,    immunofluorescence microscopy and/or scanning electron microscopy),    gene expression analysis, cytokine/chemokine secretion analysis,    metabolite analysis, polymerase chain reaction, immunoassays, ELISA,    gene arrays, or any combinations thereof-   118. The method of paragraph 116 or 117, wherein measurement of the    response of the cells or at least one component present in a fluid    within the device or present in an output fluid from the device    after exposure to the test agent determines an effect of the test    agent on the cells.-   119. The method of paragraph 118, wherein the effect comprises    ciliary clearance, cell viability, permeability of a cell layer,    cell morphology, protein expression, gene expression, cell adhesion,    adhesiveness of immune cells, cell differentiation, cytokine or    chemokine production, inflammation, or any combinations thereof.-   120. The method of paragraph 116 or 117, wherein measurement of the    response of the cells or at least one component present in a fluid    within the device or present in an output fluid from the device    after exposure to the test agent determines an efficacy of the test    agent.-   121. The method of paragraph 116 or 117, wherein measurement of the    response of the cells or at least one component present in a fluid    within the device or present in an output fluid from the device    after exposure to the test agent determines toxicity of the test    agent.-   122. The method of paragraph 116 or 117, wherein measurement of the    response of the cells or at least one component present in a fluid    within the device or present in an output fluid from the device    after exposure to the test agent determines a mechanism of efficacy    or toxicity of the test agent.-   123. The method of paragraph 116 or 117, wherein measurement of the    response of the cells or at least one component present in a fluid    within the device or present in an output fluid from the device    after exposure to the test agent determines physical-chemical,    pharmacokinetic or pharmacodynamic parameters.-   124. The method of any of paragraph 112-123, wherein when the    tissue-specific cells are adapted to be condition-specific, said    determination of the effect of the test agent identifies a    therapeutic agent for treatment of the condition.-   125. The method of any of paragraphs 112-123, wherein when the    tissue-specific cells are patient-specific, said determination of    the effect of the test agent identifies a personalized treatment for    a subject.-   126. The method of any of paragraphs 112-123, wherein when the    tissue-specific cells are patient population-specific, said    determination of the effect of the test agent identifies a treatment    specified for that particular patient subpopulation.-   127. The method of any of paragraphs 60-126, further comprising    flowing immune cells through the second sub-channel.-   128. The method of paragraph 127, wherein the tissue-specific cells    in the first sub-channel and the immune cells flowing in the second    sub-channel form an in vitro mucosal immunity model.-   129. The method of paragraph 128, wherein the mucosal immunity model    is adapted to determine efficacy or immunogenicity of a vaccine.-   130. The method of any of paragraphs 127-129, further comprising    measuring response of the immune cells.-   131. The method of paragraph 130, wherein the response of the immune    cells comprises trans-epithelial migration, maturation, activation,    cell killing, and/or drainage.-   132. The method of any of paragraphs 60-131, further comprising    connecting said at least one device to a second device comprising:    -   a second body comprising a second central channel therein; and    -   a second membrane positioned within the second central channel        and along a second plane, the second membrane configured to        separate the second central channel to form a first sub-channel        and a second sub-channel, wherein at least the first sub-channel        has a height sufficient to form a stratified structure; and    -   second tissue-specific cells on a first surface of the second        membrane facing the first sub-channel, wherein the second        tissue-specific cells display at least one characteristic        corresponding to a second pre-determined physiological endpoint.-   133. The method of paragraph 132, further comprising directing an    air flow from the first sub-channel of said at least one device to    the first sub-channel of the second device.-   134. The method of paragraph 132 or 133, wherein the tissue-specific    cells in said at least one device comprise pathogen-infected cells    and the second tissue-specific cells in the second device are normal    healthy cells.-   135. The method of any of paragraphs 132-134, further comprising    measuring response of the pathogen-infected cells upon exposure of    the air flow.-   136. The method of any of paragraphs 132-135, further comprising    measuring response of the normal healthy cells upon exposure to the    air flow from the first sub-channel of said at least one device.-   137. The method of paragraph 136, wherein the measured response of    the normal healthy cells determines transmissibility of airborne    pathogens.-   138. The method of any of paragraphs 60-137, wherein the membrane is    rigid.-   139. The method of any of paragraphs 60-137, wherein the membrane is    at least partially flexible.-   140. The method of any of paragraphs 60-139, further comprising    mechanically modulating the membrane to move or deform within the    central channel.-   141. The method of paragraph 140, wherein the mechanical modulation    of the membrane simulates a physiological strain.-   142. The method of paragraph 141, wherein the simulated    physiological strain is substantially the same as the strain    produced by motion associated with breathing, peristalsis, or heart    beating.-   143. The method of any of paragraphs 140-142, wherein the membrane    is mechanically modulated by a pneumatic mechanism.-   144. The method of paragraph 143, wherein the device further    comprises a first operating channel separated from the second    sub-channel and the first sub-channel by a first channel wall,    wherein a first edge of the membrane is fastened to the first    channel wall and a second edge of the membrane is fastened to an    opposite wall of the central channel; and wherein the first    operating channel has a first height smaller than the height of the    central channel.-   145. The method of paragraph 144, wherein the device further    comprises a second operating channel separated from the second    sub-channel and the first sub-channel by a second channel wall,    wherein the second edge of the membrane is fastened to the second    channel wall; and wherein the second operating channel has a second    height smaller than the height of the central channel.-   146. The method of paragraph 144 or 145, further comprising applying    a first pressure differential between the first operating channel    and the central channel to cause the membrane to stretch or retract    along the plane within the central channel.-   147. The method of any of paragraphs 145-146, further comprising    applying a second pressure differential between the second operating    channel and the central channel to cause the membrane to stretch or    retract along the plane within the central channel.-   148. The method of paragraph 146 or 147, wherein said applying the    first or second pressure differential comprises applying a cyclic    pressure inside the first operating channel or the second operating    channel such that the first edge of the membrane fastened to the    first channel wall and/or the second edge of the membrane fastened    to the second channel wall stretches or retracts along the plane    within the central channel.-   149. The method of paragraphs 60-148, wherein the liquid fluid in    the second sub-channel comprises a cell culture medium and/or a    biological fluid.-   150. The method of paragraph 149, wherein the biological fluid    comprises blood.-   151. A method of making a microfluidic device comprising a    microstructure and a mesostructure, wherein a dimension of the    mesostructure is substantially greater than a dimension of the    microstructure, the method comprising:    -   generating, by photolithography, a semiconductor wafer mold        having a raised feature of the microstructure; and    -   generating, by stereo-lithography, a thermoplastic mold having a        raised feature of the mesostructure.-   152. The method of paragraph 151, wherein the dimensions of the    mesostructure and the microstructure are differed by a factor of at    least 2.-   153. The method of paragraph 151 or 152, further comprising forming    the microstructure by casting in the semiconductor wafer mold.-   154. The method of any of paragraphs 151-153, further comprising    forming the mesostructure by casting in the thermoplastic mold.-   155. The method of paragraph 154, wherein the formed mesostructure    has a smooth surface finish.-   156. The method of paragraph 155, wherein the smooth surface finish    of the mesostructure facilitates bonding to the microstructure.-   157. The method of any of paragraphs 151-156, wherein the    mesostructure is a mesochannel disposed in a bottom surface of a    first substrate.-   158. The method of any of paragraphs 151-157, wherein the    microstructure is a microchannel disposed in a top surface of a    second substrate.-   159. The method of paragraph 157 or 158, further comprising placing    an at least partially porous membrane between the top surface of the    microchannel and the bottom surface of the mesochannel; and forming    a fluidic seal between the membrane and the first substrate and the    second substrate, thereby forming a body of the device having a    central channel therein, wherein the central channel comprises the    microchannel and the mesochannel separated by the membrane.-   160. The method of paragraph 159, wherein said forming the fluidic    seal comprises forming a chemical bond between the membrane and the    first substrate and the second substrate.-   161. The method of paragraph 160, wherein the chemical bond is    formed by using an adhesive chemical coating to covalently bond the    membrane to the bottom surface of first substrate and the top    surface of the second substrate.-   162. The method of paragraph 161, wherein the adhesive chemical    coating comprises (3-aminopropyl)triethoxysilane (APTES).-   163. The method of any of paragraphs 159-162, wherein the fluidic    seal is reversible such that the membrane is capable of being    removed from the device for examination.-   164. The method of paragraph 163, wherein said forming the fluidic    seal comprises clamping the membrane between the first substrate and    the second substrate together.-   165. The method of paragraph 159-164, wherein said forming the    fluidic seal comprises forming a plasma bond between the top surface    of the first substrate and the bottom surface of the second    substrate.-   166. The method of any of paragraphs 157-165, wherein the first    substrate, the second substrate, and/or the membrane comprise    polydimethylsiloxane, polyurethanes,    styrene-ethylene-butylene-styrene (SEBS), polypropylene,    polycarbonate, polyester, silicon, or any combinations thereof-   167. A method of developing a vaccine comprising:    -   providing at least one device comprising:        -   a. a body comprising a central channel therein; and        -   b. an at least partially porous membrane positioned within            the central channel and along a plane, the membrane            configured to separate the central channel to form a first            sub-channel and a second sub-channel, wherein at least the            first sub-channel has a height sufficient to form a            stratified structure; and        -   c. tissue-specific epithelial cells on a first surface of            the membrane facing the first sub-channel.    -   flowing a gaseous fluid through the first sub-channel;    -   flowing a liquid fluid comprising immune cells through the        second sub-channel;    -   contacting the tissue-specific epithelial cells with a vaccine        candidate;    -   contacting with the vaccinated tissue-specific epithelial cells        with a microbe;    -   measuring response of the tissue-specific epithelial cells to        the microbe, thereby determining efficacy of the vaccine        candidate against the microbe.-   168. The method of paragraph 167, wherein the tissue-specific    epithelial cells are contacted with the vaccine candidate at    different dosages, thereby determining an optimum dosage of the    vaccine candidate against the microbe.-   169. The method of paragraph 167 or 168, further comprising    measuring response of the immune cells.-   170. The method of paragraph 169, wherein the response of the immune    cells comprises trans-epithelial migration, maturation, activation,    cell killing, and/or drainage.-   171. The method of any of paragraphs 60-170, wherein the central    channel is linear.-   172. The method of any of paragraphs 60-170, wherein the central    channel comprise a non-linear portion.-   173. The method of any of paragraphs 144-148, wherein the height of    the first and/or second operating channel is larger than the height    of the central channel.-   174. The method of any of paragraphs 144-148, wherein the height of    the first and/or second operating channels is substantially the same    as or smaller than the height of the central channel.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±5%.

In one aspect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising”). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1. Differentiation of Human Primary Airway (e.g., Small Airway)Epithelial Cells into Ciliated, Mucous-Secreting and Clara Cells

Methods of differentiating airway epithelial cells in transwell systemshas been previously described (Villenave et al., PNAS 2012, 109 (13)5040-5045; and Villenave et al., J. Virol., 2010, 84 (22) 11718-11728).However, technologies and methods for differentiating primary humanairway epithelial cells in microfluidic setting have not yet existed.Presented below is an example protocol to differentiate human primaryairway epithelial cells (e.g., primary small airway epithelial cells).

Medium #1

Bronchial Epithelial cell Basal Medium (BEBM) 500 ml (Lonza; Cat #CC-3171) plus BEGM SingleQuot Kit Suppl. & Growth Factors (Lonza; Cat #CC-4175); these supplements are shown as follows:

-   -   1. BPE, ˜2 ml    -   2. Hydrocortisone, ˜0.5 ml    -   3. hEGF (human epidermal growth factor), 0.5 ml    -   4. Epinephrine, ˜0.5 ml    -   5. Transferrin, ˜0.5 ml    -   6. Insulin, ˜0.5 ml    -   7. Retinoic Acid, ˜0.5 ml    -   8. Triiodothyronine (T3), ˜0.5 ml    -   9. GA-1000 (gentamicin), ˜0.5 ml        Medium #2

BEBM 250 ml (Lonza; Cat # CC-3171) plus DMEM 250 ml (Life Technologies,Cat #11885-092)—supplemented with 1% v/v penicillin/streptomycin, plusthe following supplements & growth factors:

-   -   1. BPE, ˜2 ml    -   2. Hydrocortisone, ˜0.5 ml    -   3. hEGF (human epidermal growth factor), ˜0.5 ml    -   4. Epinephrine, ˜0.5 ml    -   5. Transferrin, ˜0.5 ml    -   6. Insulin, ˜0.5 ml    -   7. BSA (bovine serum albumin), ˜1 ml of ˜1.5 mg/ml per ˜500 ml        BEBM/DMEM mixed medium    -   8. Retinoic Acid, ˜0.5 ml of ˜0.015 mg/ml per ˜500 ml BEBM/DMEM        mixed medium

Items 1-6 were obtained from BEGM SingleQuot Kit Suppl. & Growth Factors(Lonza; Cat # CC-4175). Item 7 was purchased from Sigma, Cat # A9576,and diluted in Medium #1. Item 8 was purchased from Sigma, Cat # R2625,and diluted in DMSO

Medium #3

BEBM 250 ml (Lonza; Cat # CC-3171) plus DMEM 250 ml (Life Technologies,Cat #11885-092) plus the following supplements & growth factors:

-   -   1. BPE, ˜2 ml    -   2. Hydrocortisone, ˜0.5 ml    -   3. hEGF (human epidermal growth factor), ˜0.5 ml    -   4. Epinephrine, ˜0.5 ml    -   5. Transferrin, ˜0.5 ml    -   6. Insulin, ˜0.5 ml    -   7. BSA (bovine serum albumin), ˜1 ml of ˜1.5 mg/ml per ˜500 ml        BEBM/DMEM mixed medium    -   8. Retinoic Acid, ˜0.5 ml of ˜3 mg/ml per ˜500 ml BEBM/DMEM        mixed medium

Items 1-6 were obtained from BEGM SingleQuot Kit Suppl. & Growth Factors(Lonza; Cat # CC-4175). Item 7 was purchased from Sigma, Cat # A9576,and diluted in Medium #1. Item 8 was purchased from Sigma, Cat # R2625,and diluted in DMSO.

Differentiation Procedure

-   -   About 2×10⁵ primary airway epithelial cells (e.g., primary small        airway epithelial cells) re-suspended in about 20-40 μl of        medium #2 is added through inlet or outlet of “airway lumen”        channel into the device according to one embodiment as shown in        FIG. 2G;    -   Cells are incubated for at least about 3 hours at 37° C., 5% CO₂        to allow adhesion to the membrane (e.g., the rigid polyester or        polycarbonate membrane or flexible PDMS membrane);    -   Device is connected to medium flow on both top mesochannel and        bottom microchannel at about 30 μl/h using syringe pumps or        about 61 μl/h using peristaltic pump;    -   Medium #2 is used for culture under submerged condition    -   About 4-5 days post-culture when cells reach full confluence, an        air-liquid interface (ALI) is generated by removing the apical        medium slowly through the outlet of the “airway lumen” channel;    -   Medium #3 is used for culture during ALI;    -   Cells are kept in culture for about 3-4 weeks with periodic        replenishment of fresh medium (e.g., freshly prepared media is        added to a reservoir every 5-7 days);    -   The quality of differentiated chips is assessed regularly by        microscopic imaging. In some embodiments, the device can be        disconnected from the flow. About ˜400-200 μl medium #3 is added        into the “airway lumen” channel and the device is visualized        under a microscope—after examination, the apical medium (in the        “airway lumen” channel) is removed to restore ALI until the        cells become fully differentiated.

The differentiated state of the cells can be determined by detecting thepresence of cilia-associated markers, globet cell-associated markers,and/or tight junction-associated markers. For example, FIG. 5D is a setof immunofluorescence images showing formation of a primary small airwayepithelium on the membrane. Tight junction proteins (e.g., TJP-1 and/orZO-1) were detected to indicate a functional tight junction barrierformed by the formed epithelium. FIG. 5E is a set of immunofluorescenceand SEM images showing differentiation of the airway epithelial cells tociliated cells. FIG. 5F shows a 3D view of differentiated epithelialprimary cells (cilia beating: detected by beta-tubulin IC; and mucussecretion: detected by Muc5AC) in the device described herein. FIG. 5Gshows representative images of ciliated cells along the length of themesochannel of the device described herein. A uniform distribution ofabundant cilia beating after about 3 weeks of culturing at an air-liquidinterface is a hallmark of epithelial differentiation in vivo.

Using the devices described herein, the small airway human epithelial(bronchiolar) cells can also be differentiated into Clara cells. Thesecells are apically dome-shaped cells that contain drug-metabolizingenzymes like p450 and secrete proteins like CC10 (Clara cell secretoryprotein 10). An antibody against CC10 was used to identify these cellsin the device. FIG. 29A is a confocal microscopic top view image ofClara cells stained for CC10 and ciliated cells labeled with β-tubulinIV following well-differentiation of bronchiolar cells in the device.The Clara cells were also imaged with scanning electron microscopy. FIG.29B is a scanning electron micrograph of differentiated bronchiolarcells grown in the device, showing the extensive ciliated cells coverage(“1” arrow), microvilli (“2” arrow) that normally indicates apicalmembrane of mucous-producing goblet cells, and some dome-shapedstructures that indicate Clara cells (“3” arrow).

Example 2. Uses of the Devices Described Herein to Mimic Effects ofChronic Obstructive Pulmonary Disease (COPD)

Similar differentiation protocol as described in Example 1 above can beused to culture airway epithelial cells obtained from both healthynormal and chronic obstructive pulmonary disease (COPD) airways andinduce the cells to differentiate into pseudostratified mucociliaryepithelium in the devices described herein. FIGS. 25A-25B are confocalimages of well-differentiated normal and COPD epithelia followingair-liquid interface (ALI) induction in the devices described herein.

To determine if the COPD disease phenotypes were established in thedevices described herein, the differentiated cells were stimulated,e.g., with a pro-inflammatory agent (e.g., a pathogen or fragmentsthereof such as lipopolysaccharides) and gene expression levels ofToll-like receptor 4 (TLR4) and TLR3 were then measured and compared tothe levels in the normal cells.

TLRs, e.g., TLR3 and TLR4, are molecules that mediate recognition andresponse to stimulants (e.g., pathogens or fragments thereof such aslipopolysaccharides) in airway epithelial cells. A quantitative assay(e.g., real-time polymerase chain reaction (qPCR)) was performed toidentify and determine difference in mRNA levels of TLR4 and TLR3expression between normal/healthy and COPD-derived epithelial cells thatwere grown in the devices described herein. FIG. 26A shows that COPDdevices (i.e., devices with COPD-derived cells) exhibited lower TLR3 andTLR4 mRNA levels than healthy devices (i.e., devices with healthycells). The difference in TLR4 mRNA levels detected between the COPDdevices and healthy devices is consistent with what is generallyobserved between COPD and healthy patients.

It was next sought to determine cytokine production in response to TLRstimulation. Cytokines are molecules that are involved in inflammationand their generation can be modulated by TLR activation. After thewell-differentiated (i.e. mucociliary) epithelium (normal andCOPD-derived) was stimulated with lipopolysaccharides (LPS), a qPCR wasperformed on the cells to compare inflammatory response between COPD andhealthy epithelia in the devices described herein. LPS is a bacterialderived molecule that stimulates TLR4. It was found that LPS selectivelyinduced IL8 secretion from COPD epithelial cells without increasing IL8in healthy epithelial cells (FIG. 26B). This is in line with clinicalreports and other ex vivo observations that COPD patients arehyper-reactive inflammation-wise and produce more pro-inflammatorymediators. Similarly, when the differentiated (i.e. mucociliary)epithelium (normal and COPD-derived) was stimulated with poly (I:C)(polyinosinic:polycytidylic acid—a synthetic analogue of thedouble-stranded RNA that mimic viral infection and stimulates TLR3),poly(I:C) selectively up-regulated M-CSF in COPD epithelial cells whilethere was no significant change in healthy epithelial cells (FIG. 26C).M-CSF is a cytokine that promotes survival and differentiations of asubset of immune cells, e.g., macrophages, in our bodies. This agreeswith general observations that in the lungs of COPD patients there isgenerally an increased number of macrophages as compared to the numberof macrophages in healthy individuals. The expression of two othercytokines (IP-10 and RANTES) was induced in both healthy donor and COPDepithelial cells by poly (I:C) (FIGS. 26D-26E).

Example 3. Establishment of a Complex 3-Cell Type MicrofluidicCo-Culture System

Any embodiment of the devices described herein can be used to establisha 3-cell type microfluidic co-culture system as described below. The3-cell type microfluidic co-culture system comprises ciliatedepithelium, endothelium and circulating leukocytes. By way of exampleonly, FIG. 27A shows one embodiment of the devices that was used in thisExample. The device comprised two parallel channels separated by anECM-coated porous membrane: (i) a top mesochannel (“airway lumen”channel) with height corresponding to radius of a human lung smallairway (1000 μm) and (ii) a bottom microchannel (“microvascular”channel) (100 μm deep) to re-create post-capillary venules (major sitesof leukocyte recruitment and adhesion in vivo). The epithelium wascultured on one side of the membrane facing the mesochannel, while theendothelium was cultured on another side of the membrane facing themicrochannel. Neurophils, a subset of circulating immune cells importantin infection and inflammation and accumulate in and contribute to lungpathology in many diseases including COPD, were then introduced into the“microvascular” channel.

To visualize endothelium-leukocyte interactions, differentiated airwayepithelial cells were stimulated in the device with poly (I:C) 10 μg/mlfor 6 h to mimic inflammation phenotype by TLR3 pathway stimulation.Freshly isolated blood neutrophils were then flowed over an endotheliallayer (activated by the stimulation of the airway epithelial cells withpoly (I:C)) through “microvascular” channel to generate physiologicalwall shear stress of 1 dyne/cm². A series of time-lapse microscopicimages (FIG. 27B) showed capture of a flowing neutrophil (not visible inthe first panel from left but appears in the second panel; shown by thearrow head) to endothelium adjacent to a pre-adhered neutrophil(circles). Following initial attachment the neutrophil crawled overapical surface of activated endothelium and then firmly adhered (timesindicated in seconds). The shadows in background are weak endothelialflorescence signals bleeding into neutrophil channel during livehigh-speed multichannel image acquisition.

To analyze cell adhesion molecules gene expression, fully differentiatedepithelial cells were co-cultured in the device with pulmonarymicrovascular endothelial cells, stimulated apically with poly (I:C) 10μg/ml for 6 h and endothelial cells were then lysed to determineE-selectin and VCAM1 mRNA levels. E-selectin (endothelial selectin) is acell adhesion molecule expressed on endothelial cells and up-regulatedduring inflammation. VCAM1 (vascular cell adhesion molecule 1) isanother cell adhesion molecule that endothelial cells express. Both ofthese molecules are important for capture and adhesion of leukocytesfrom circulation. As shown in FIG. 27C, epithelial challenge with poly(I:C) induced a significant up-regulation of E-selectin gene expressionand a higher, but not statistically significant, VCAM-1 transcriptlevels in endothelial cells, as compared to the cells without the poly(I:C) challenge.

Example 4. Comparing Drug Efficacy on Neutrophil Capture and Adhesionand Inflammation Suppression in a Small Airway Mimicking Device

COPD epithelium-microvascular endothelium co-culture devices wereestablished, e.g., as described in Example 1 or 2, and then were eithertreated with the corticosteroid drug budesonide (10 nM) or PFI-2 (abromodomain-containing protein 4 (BRD4) inhibitor drug; 500 nM), or leftuntreated. The COPD epithelium was then stimulated with poly (I:C) 10μg/ml via the “airway lumen” channel for 6 h and then examined foradhesion of recruited neutrophils. Neutrophils were stained with Hoechstimmediately prior to experiment to allow visualization andquantification. Representative immunofluorescence images of the threeconditions are illustrated in FIG. 28A.

Effects of budesonide and PFI-2 on neutrophil adhesion were quantified.PFI-2 significantly lowered neutrophil recruitment compared with notreatment and budesonide, whereas the effect of budesonide was notsignificant (FIG. 28B).

To compare the ability of budesonide and PFI-2 in suppressinginflammatory cytokine secretion, secreted cytokines from the“microvascular” channel of the co-culture devices were also analyzed byMultiplex Cytokine Detection System prior to neutrophil recruitmentassay. FIG. 28C shows that PFI-2, unlike budesonide, significantlylowered secretion of neutrophil-chemoattractants IL-8, GROα and GM-CSF,monocyte-chemoattractant MCP-1, and acute inflammation associatedcytokine IL-6.

Example 5. Induction of Asthma-Like Phenotype in the Airway-On-A-Chipfor Assessment of Drug Efficacy

To develop asthma-like phenotype in the devices described herein, airwaycells were differentiated in the devices, e.g., using the methods asdescribed in Example 1 or 2, and then stimulated with IL-13 to induceasthma-like phenotype in the devices. IL-13 is a protein secreted byimmune cells which is found in high quantities in lungs of asthmatics.The cells in the devices stimulated with IL-13 reproduced at least fewhallmarks of asthma, e.g., with a higher number of goblet cells (cellsthat produce mucus) (FIGS. 30A and 30B), lower cilia beating frequency(FIG. 30D) and higher secretion of G-CSF and GM-CSF (FIG. 30C), ascompared to cells without IL-13 stimulation.

The “airway” devices were then used to assess the drug efficacy ofTofacitinib, a JAK inhibitor. The IL-13 stimulated cells were treatedwith Tofacitinib and it was found that the drug was able to reversephenotypes associated with asthma in the IL-13 stimulated cultures. Forexample, the drug was able to decrease the number of goblet cells (FIGS.30A-30B), to decrease GM-CSF and G-CSF secretion (FIG. 30C), and/or alsoto increase cilia beating frequency (FIG. 30D), in IL-13 stimulatedcultures to healthy levels.

What is claimed is:
 1. A method for culturing chronic obstructivepulmonary disease (COPD) cells comprising: 1) providing a microfluidicdevice comprising: a) a body comprising a central channel therein; andb) an at least partially porous membrane positioned within the centralchannel, wherein the at least partially porous membrane separates atleast one microchannel and at least one mesochannel, wherein a heightratio of the at least one mesochannel to the at least one microchannelranges from about 1.1:1 to about 50:1, wherein said at least onemesochannel comprises a liquid; 2) seeding human epithelial cells from aCOPD patient on said membrane facing the at least one mesochannel; 3)culturing the seeded human cells from step 2) on said membrane submergedwithin a first liquid; 4) removing the first liquid from the at leastone mesochannel, such that a gas-liquid interface is established,whereby said human cells are induced to differentiate intopseudostratified epithelial cells.
 2. The method of claim 1, wherein thehuman epithelial cells are primary cells.
 3. The method of claim 1,wherein the COPD epithelial cells are selected from the group consistingof airway cells, bronchial cells, and nasal epithelial cells.
 4. Themethod of claim 1, wherein the cells are from a patient and the patienthas a disease selected from the group consisting of asthma, cysticfibrosis, sarcoidosis, and idiopathic lung fibrosis.
 5. The method ofclaim 1, wherein the at least partially porous membrane is positionedalong a plane and is configured to separate the central channel to forma first channel and a second channel, and at least the first channel hasa height sufficient to form a stratified structure.
 6. A devicecomprising: a) a body comprising a central channel therein; and b) an atleast partially porous membrane positioned within the central channel,the membrane configured to separate the central channel to form at leastone microchannel and at least one mesochannel, wherein a height ratio ofthe at least one mesochannel to the at least one microchannel rangesfrom about 1.1:1 to about 50:1.
 7. The device of claim 6, wherein theheight ratio of the mesochannel to the microchannel ranges from about2.5:1 to about 50:1.
 8. The device of claim 6, wherein the height ratioof the mesochannel to the microchannel ranges from about 5:1 to about25:1.
 9. The device of claim 6, wherein the membrane is rigid.
 10. Thedevice of claim 6, wherein the membrane is at least partially flexible.11. The device of claim 6, wherein the membrane has a thickness of about100 nm to about 50 μm.
 12. The device of claim 6, wherein said pores ofthe membrane has a diameter of about 0.1 μm to about 15 μm.
 13. Thedevice of claim 6, wherein center-to-center spacing between said poresranges from about 1 μm to about 100 μm.
 14. The device of claim 6,wherein at least one surface of the membrane comprises cells adhered tothereto.